Selective estrogen receptor degraders

ABSTRACT

pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof, wherein either R1 or R2 is independently selected from Cl, F, —CF3, or —CH3, and the other is hydrogen, and methods for their use are provided.

This application claims the benefit of U.S. Provisional Application No.62/697,100, filed Jul. 12, 2018.

BACKGROUND

Selective estrogen receptor degraders (SERDs) bind to the estrogenreceptor (ER) and downregulate ER-mediated transcriptional activity.This degradation and downregulation caused by SERDs can be useful in thetreatment of cell proliferation disorders, such as cancer. Some smallmolecule examples of SERDs have been disclosed in the literature (see,e.g., WO2005073204, WO2014205136, and WO2016097071). However, knownSERDs have not yet been as useful as is needed to effectively treatcancer. For example, finding SERDs with better pharmacokinetic (PK) andpharmacodynamic (PD) properties, higher efficiency in the clinic, andgood oral bioavailability would be very helpful in treating cancer. Apure antagonist SERD with potent inhibition of ER-mediated transcriptionwould be expressly beneficial in treating cancer. There is a need fornew SERDs to treat cancers such as breast cancer, ovarian cancer,endometrial cancer, prostate cancer, uterine cancer, gastric cancer, andlung cancer as well as mutations due to emerging resistance. Inparticular there is a need for new SERDs to treat ER-positive breastcancer, gastric cancer, and/or lung cancer.

SUMMARY

Compounds of the formula:

and pharmaceutically acceptable salts thereof, and pharmaceuticalcompositions thereof, are provided herein. In this formula either R¹ orR² is independently selected from Cl, F, —CF₃, or —CH₃, and the other ishydrogen.

Methods of using the compounds as described herein, pharmaceuticallyacceptable salts thereof, and pharmaceutical compositions thereof, totreat breast cancer, ovarian cancer, endometrial cancer, prostatecancer, uterine cancer, gastric cancer, or lung cancer are alsoprovided. The methods include administering a therapeutically effectiveamount of a compound as described herein, or a pharmaceuticallyacceptable salt thereof, to a patient in need.

Further provided are the compound as described herein, and apharmaceutically acceptable salts thereof, for use in therapy. Thecompounds described herein, and pharmaceutically acceptable saltsthereof, can be used in the treatment of breast cancer, ovarian cancer,endometrial cancer, prostate cancer, uterine cancer, gastric cancer, orlung cancer.

The use of the compounds as described herein, and pharmaceuticallyacceptable salts thereof, for the manufacture of a medicament fortreating breast cancer, ovarian cancer, endometrial cancer, prostatecancer, uterine cancer, gastric cancer, or lung cancer is furtherprovided.

DETAILED DESCRIPTION

Novel tetracyclic compounds and pharmaceutical salts thereof that act asSERDs are disclosed herein. The newly invented SERDs that are describedherein provide inhibition of ER-mediated transcription that will beuseful in treating cancers such as breast cancer, ovarian cancer,endometrial cancer, prostate cancer, uterine cancer, gastric cancer, andlung cancer as well as mutations due to emerging resistance. These SERDscan be used either as single agents or in combination with other classesof drugs including selective estrogen receptor modulators (SERMs),aromatase inhibitors, CDK4 inhibitors, CDK6 inhibitors, PI3K inhibitors,and mTOR inhibitors to treat hormone receptor-positive cancers such asbreast cancer, gastric cancer, and/or lung cancer.

The novel compounds described herein are represented by Formula I:

and pharmaceutically acceptable salts thereof, wherein either R¹ or R²is independently selected from Cl, F, —CF₃, or —CH₃, and the other ishydrogen. One of skill in the art will appreciate that compounds asdescribed by Formula I, or pharmaceutically acceptable salts thereof,contain a chiral center, the position of which is indicated by an *above. One of skill in the art will also appreciate that theCahn-Ingold-Prelog (R) or (S) designations for chiral centers will varydepending upon the substitution patterns around a chiral center. Thechiral center in the compound of Formula I provides an R-enantiomericform shown by Formula II:

And an S-enantiomeric form shown by Formula III:

All individual stereoisomers, enantiomers, and diastereomers, as well asmixtures of the enantiomers and diastereomers of the compounds accordingto Formula I, Formula II, and Formula III including racemates areincluded within the scope of the compounds described herein. Compoundsfor pharmaceutical use that contain chiral centers are often isolated assingle enantiomers or diastereomers and such isolated compounds ofFormula I, Formula II, and Formula III are included within the scope ofthe compounds disclosed herein. One of skill in the art will alsoappreciate that the compounds of Formula I, Formula II, and Formula IIIdescribed herein, and pharmaceutically acceptable salts thereof, can bedeuterated (where a hydrogen can be replaced by a deuterium) and suchmolecules are considered to be included within the scope of thecompounds disclosed herein.

Specific examples of the compounds of Formula I (including IUPACnomenclature names) are shown here:

5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol

5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-7-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol

8-chloro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol

7-chloro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol

8-fluoro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol

7-fluoro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol

5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-methyl-5H-[1]benzopyrano[4,3-c]quinolin-2-ol;and

5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-7-methyl-5H-[1]benzopyrano[4,3-c]quinolin-2-ol

Due to the chiral center noted above, each of these specific examples ofcompounds of Formula I shown above have R- and S-enantiomeric forms(i.e., R-enantiomeric compounds of Formula II and S-enantiomericcompounds of Formula III) as shown in Table 1.

TABLE 1 Enantiomeric forms of compounds of Formula I Chemical NameR-enantiomer (Formula II) S-enantiomer (Formula III) 5-(4-{2-[3-(fluoromethyl)azetidin- 1-yl]ethoxy}phenyl)- 8-(trifluoromethyl)- 5H-[1]benzopyrano[4,3- c]quinolin-2-ol

5-(4-{2-[3- (fluoromethyl) azetidin-1- yl]ethoxy}phenyl)-7-(trifluoromethyl)-5H- [1]benzopyrano[4,3- c]quinolin-2-ol

8-chloro-5-(4-{2- [3-(fluoromethyl) azetidin-1- yl]ethoxy}phenyl)-5H-[1] benzopyrano[4,3- c]quinolin-2-ol

7-chloro-5-(4-{2-[3- (fluoromethyl) azetidin-1- yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3- c]quinolin-2-ol

8-fluoro-5-(4-{2-[3- (fluoromethyl) azetidin-1- yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3- c]quinolin-2-ol

7-fluoro-5-(4-{2-[3- (fluoromethyl) azetidin-1- yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3- c]quinolin-2-ol

5-(4-{2-[3- (fluoromethyl) azetidin-1- yl]ethoxy}phenyl)- 8-methyl-5H-[1]benzopyrano[4,3- c]quinolin-2-ol

5-(4-{2-[3- (fluoromethyl) azetidin-1- yl]ethoxy}phenyl)- 7-methyl-5H-[1]benzopyrano[4,3- c]quinolin-2-ol

Also described herein are pharmaceutical compositions including thecompounds of Formula I, Formula II, and Formula III as described herein,or pharmaceutically acceptable salts thereof, in combination with apharmaceutically acceptable excipient, carrier, or diluent. Thepharmaceutical compositions described herein may be prepared usingpharmaceutically acceptable additives. The term “pharmaceuticallyacceptable additive(s)” as used herein, refers to one or more carriers,diluents, and excipients that are compatible with the other additives ofthe compositions or formulations and not deleterious to the patient. Thecompounds of Formula I, Formula II, and Formula III, or pharmaceuticallyacceptable salts thereof, described herein can be formulated aspharmaceutical compositions administered by a variety of routes, such asoral or IV. Bioavailability is often a factor in cancer treatment andthe ability to choose administration methods and pharmaceuticalcompositions to control or optimize the bioavailability of an activeingredient is useful. For example, an orally bioavailable SERDcomposition would be particularly useful. The compounds of Formula I,Formula II, and Formula III, or pharmaceutically acceptable saltsthereof, as described herein are believed to have oral bioavailability.Examples of pharmaceutical compositions and processes for theirpreparation can be found in “Remington: The Science and Practice ofPharmacy”, L. V. Allen Jr, Editor, 22nd Ed., Mack Publishing Co., 2012.Non-limiting examples of pharmaceutically acceptable carriers, diluents,and excipients include the following: saline, water, starch, sugars,mannitol, and silica derivatives; binding agents such as carboxymethylcellulose and other cellulose derivatives, alginates, gelatin, andpolyvinyl-pyrrolidone; kaolin and bentonite; and polyethyl glycols.

Further described herein are methods of treating a cancer. The methodsdescribed herein include administering to a patient in need of suchtreatment an effective amount of a compound of Formula I, Formula II,and Formula III as described herein, or a pharmaceutically acceptablesalt thereof. For example, the method of administering the effectiveamount of a compound of Formula I, Formula II, and Formula III asdescribed herein, or a pharmaceutically acceptable salt thereof, can beoral administration. The cancer can be an estrogen responsive cancer.Additionally, the cancer can be breast cancer, ovarian cancer,endometrial cancer, prostate cancer, uterine cancer, gastric cancer, orlung cancer. For example, the cancer can be ER-positive breast cancer,ER-positive gastric cancer, or ER-positive lung cancer.

Also described herein are compounds of Formula I, Formula II, andFormula III as described herein, or pharmaceutically acceptable saltsthereof, for use in therapy. Also provided herein are the compounds ofFormula I, Formula II, and Formula III as described herein, orpharmaceutically acceptable salts thereof, for use in the treatment ofbreast cancer, ovarian cancer, endometrial cancer, prostate cancer,uterine cancer, gastric cancer, or lung cancer. In particular the breastcancer can be ER-positive breast cancer, ER-positive gastric cancer, orER-positive lung cancer. For example, the compound of Formula I, FormulaII, and Formula III, or pharmaceutically acceptable salt thereof, can beorally administered.

Additionally, the compounds of Formula I, Formula II, and Formula III asdescribed herein, or pharmaceutically acceptable salts thereof, can beused in the manufacture of a medicament for the treatment of a cancer.For example, the medicament can be orally administered. The types ofcancer the medicaments as described herein can be used to treat includebreast cancer, ovarian cancer, endometrial cancer, prostate cancer,uterine cancer, gastric cancer, or lung cancer. In particular the cancercan be ER-positive breast cancer, ER-positive gastric cancer, orER-positive lung cancer.

The compounds of Formula I, Formula II, and Formula III as describedherein, and pharmaceutically acceptable salts thereof, may have clinicalutility as a single agent or in combination with one or more othertherapeutic agents (e.g., anti-cancer agents), for the treatment ofcancers such as breast cancer, ovarian cancer, endometrial cancer,prostate cancer, uterine cancer, gastric cancer, or lung cancer. Whenused in combination with other therapeutic agents (such as anti-canceragents), the compounds of Formula I, Formula II, and Formula III asdescribed herein, or pharmaceutically acceptable salts thereof, can beused simultaneously, sequentially, or separately with other therapeuticagents. Examples of classes of drugs that the compounds of Formula I,Formula II, and Formula III as described herein, or pharmaceuticallyacceptable salts thereof, can be combined with include SERMs, aromataseinhibitors, CDK4 inhibitors, CDK6 inhibitors, PI3K inhibitors, and mTORinhibitors to treat hormone receptor-positive breast cancer. Morespecific examples of drugs with which the compounds of Formula I,Formula II, and Formula III as described herein, or pharmaceuticallyacceptable salts thereof, can be combined include abemaciclib (CDK4/6inhibitor), everolimus (mTOR inhibitor), alpelisib (PIK3CA inhibitor),and 8-[5-(1-hydroxy-1-methylethyl)pyridin-3-yl]-1-[(2S)-2-methoxypropyl]-3-methyl-1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one(PI3K/mTOR inhibitor).

As used herein, the term “effective amount” refers to the amount or doseof a compound of Formula I, Formula II, and Formula III as describedherein, or a pharmaceutically acceptable salt thereof, which, uponsingle or multiple dose administration to the patient, provides thedesired effect in the patient under diagnosis or treatment. Preferably,a desired effect is inhibition of tumor cell proliferation, tumor celldeath, or both. The compounds of Formula I, Formula II, and Formula IIIas described herein, or pharmaceutically acceptable salts thereof, aregenerally effective over a wide dosage range. For example, dosages perday normally fall within the daily range of about 100 mg to about 2000mg.

As used herein, “treat”, “treating” or “treatment” refers torestraining, slowing, stopping, or reversing the progression or severityof an existing symptom or disorder.

As used herein, the term “patient” refers to a human which is afflictedwith a particular disease, disorder, or condition.

The compounds of Formula I, Formula II, and Formula III as describedherein, or pharmaceutically acceptable salts thereof, may be prepared bya variety of procedures known in the art, some of which are illustratedin the Preparations and Examples below. The specific synthetic steps foreach of the routes described may be combined in different ways, or inconjunction with steps from different procedures, to prepare compoundsof Formula I, Formula II, and Formula III as described herein, orpharmaceutically acceptable salts thereof. The products can be recoveredby conventional methods well known in the art, including extraction,evaporation, precipitation, chromatography, filtration, trituration, andcrystallization. The reagents and starting materials are readilyavailable to one of ordinary skill in the art.

Intermediates and processes useful for the synthesis of the compounds ofFormula I, Formula II, and Formula III as described herein are intendedto be included in this description. Additionally, certain intermediatesdescribed herein may contain one or more protecting groups. The variableprotecting group may be the same or different in each occurrencedepending on the particular reaction conditions and the particulartransformations to be performed. The protection and deprotectionconditions are well known to the skilled artisan and are described inthe literature (See for example “Greene's Protective Groups in OrganicSynthesis”, Fourth Edition, by Peter G. M. Wuts and Theodora W. Greene,John Wiley and Sons, Inc. 2007).

Individual isomers, enantiomers, and diastereomers may be separated orresolved by one of ordinary skill in the art at any convenient point inthe synthesis of compounds of Formula I, Formula II, and Formula III asdescribed herein, by methods such as selective crystallizationtechniques or chiral chromatography (See for example, J. Jacques, etal., “Enantiomers, Racemates, and Resolutions”, John Wiley and Sons,Inc., 1981, and E. L. Eliel and S. H. Wilen, “Stereochemistry of OrganicCompounds”, Wiley-Interscience, 1994). While individual isomers,enantiomers, and diastereomers may be separated or resolved as noted,their Cahn-Ingold-Prelog (R) or (S) designations for chiral centers maynot yet have been determined. Where Cahn-Ingold-Prelog (R) or (S)designations are not available, the identifiers “isomer 1” and “isomer2” are used and are combined with the IUPAC name withoutCahn-Ingold-Prelog stereochemistry designation. The compounds of FormulaI, Formula II, and Formula III being identified as “isomer 1” or “isomer2” herein are isolated as defined in the specific experimentaldescriptions below. Whether an isomer is a “1” or a “2” refers to theorder in which the compounds of Formula I, Formula II, and Formula IIIelute from a chiral chromatography column, under the conditions listed,i.e., an “isomer 1” is the first to elute from the column under thenoted conditions. If chiral chromatography is initiated early in thesynthesis, the same designation is applied to subsequent intermediatesand compounds of Formula I, Formula II, and Formula III.

Unless specifically noted, abbreviations used herein are definedaccording to Aldrichimica Acta, Vol. 17, No. 1, 1984. Otherabbreviations are defined as follows: “ACN” refers to acetonitrile;“BSA” refers to Bovine Serum Albumin; “cataCXium® A Pd G3” refers to[(di(1-adamantyl)-butylphosphine)-2-(2′-amino-1,1′-biphenyl)]palladium(II)methanesulfonate; “DCM” refers to dichloromethane or methylene chloride;“DMA” refers to dimethylacetamide; “DMEA” refers to dimethylethylamine;“DMEM” refers to Dulbecco's Modified Eagle's Medium; “DMF” refers toN,N-dimethylformamide; “DMSO” refers to dimethyl sulfoxide; “DNA” refersto deoxyribonucleic acid; “cDNA” refers to complementary DNA; “DNase”refers to deoxyribonuclease; “DTT” refers to dithiothreitol; “EC₅₀”refers to the concentration of an agent which produces 50% response ofthe target activity compared to a predefined positive control compound(absolute EC₅₀); “EDTA” refers to ethylenediaminetetraacetic acid; “ee”refers to enantiomeric excess; “ERa” refers to estrogen receptor alpha;“ERβ” refers to estrogen receptor beta; “EtOAc” refers to ethyl acetate;“EtOH” refers to ethanol or ethyl alcohol; “FBS” refers to Fetal BovineSerum; “HBSS” refers to Hank's Balanced Salt Solution; “HEC” refers tohydroxy ethyl cellulose; “HEPES” refers to4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; “HPLC” refers tohigh-performance liquid chromatography; “IC₅₀” refers to theconcentration of an agent which produces 50% of the maximal inhibitoryresponse possible for that agent, (relative IC₅₀), or the concentrationof an agent which produces 50% inhibition of the target enzyme activitycompared to placebo control (absolute IC₅₀); “IPA” refers toisopropylamine; “iPrOH” refers to isopropanol or isopropyl alcohol; “IV”refers to intravenous administration; “K_(i)” refers to inhibitionconstant; “MEK” refers to methylethyl ketone; “MeOH” refers to methylalcohol or methanol; “MTBE” refers to methyl t-butyl ether; “PBS” refersto Phosphate Buffered Saline; “PO” refers to oral administration; “PRa”refers to progesterone receptor alpha; “QD” refers to once a day dosing;“RNA” refers to ribonucleic acid; “RNase” refers to ribonuclease;“RT-PCR” refers to reverse transcription polymerase chain reaction;“RT-qPCR” refers to reverse transcription quantitative polymerase chainreaction; “SFC” refers to supercritical fluid chromatography; “TED₅₀”refers to the effective dose to achieve 50% inhibition of the target inthe tumors; “THF” refers to tetrahydrofuran; “t_((R))” refers toretention time; “XantPhos Pd G2” refers tochloro[(4,5-bis(diphenylphosphino)-9,9-dimethylxanthene)-2-(2′-amino-1,1′-biphenyl)]palladium(II);and “XPhos Pd G2” refers tochloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II).

The following preparations and examples further illustrate theinvention.

PREPARATIONS AND EXAMPLES

Scheme 1 depicts the synthesis of compounds of Formula I.

In Step A, a Grignard reaction is accomplished. A Grignard reaction iswell known in the art as a reaction for the formation of carbon-carbonbonds. The reaction involves an organometallic reaction in which an arylmagnesium halide, the Grignard reagent adds to a carbonyl group such asthe acid chloride of compound 2 to give the compound of Step A. Forexample, a 4-chloro-substituted quinolone, compound 1, is treated with aGrignard reagent such as isopropylmagnesium chloride to form a Grignardintermediate followed by the addition of an acid chloride,4-fluorobenzoyl chloride, compound 2, in a solvent such as THF. Atcompletion, the reaction can be quenched with water to give compound 3.

In Step B, the aryl methyl ether of compound 3 may be demethylated undera variety of conditions recognizable to the skilled artisan such astreatment with boron tribromide. For example, compound 3 is slowlytreated with boron tribromide at a temperature of about 0° C. in asolvent such as DCM. The mixture is stirred at room temperature andquenched with dibasic potassium phosphate to give compound 4.

In Step C, the azetidine ether 6 may be formed by treatment of thecorresponding p-fluorophenyl ketone 4 and the azetidine alcohol salt 5,or the corresponding free base with a suitable base, for example sodiumhydride, sodium t-butoxide or potassium t-butoxide, in the appropriatepolar aprotic solvent such as DMF or THF to give the ether compound 6.

Compound 6 is then alkylated with the appropriate substituted arylboronic acid, compound 7, in a Suzuki cross coupling reaction to givecompound 8 in Step D. The skilled artisan will recognize that there area variety of conditions that may be useful for facilitating suchcross-coupling reactions. Suitable palladium reagents may includeXantPhos Pd G2, cataCXium® A Pd G3, bis(triphenylphosphine)palladium(II)chloride, tris(dibenzylideneacetone)dipalladium (0) withtricyclohexylphosphine,(1,1′-bis(diphenylphosphino)ferrocene)palladium(II) chloride, palladiumtetrakistriphenylphosphine, or palladium(II) acetate. Suitable bases mayinclude potassium fluoride, cesium carbonate, sodium carbonate,potassium carbonate, lithium t-butoxide, or potassium phosphate tribasicmonohydrate. Compound 6, for example, can be reacted with theappropriate boronic acid, compound 7, such as2-fluoro-4-(trifluoromethyl)phenylboronic acid in a solvent such as2-methyl-2-butanol with a base such as potassium carbonate and acatalyst such as XPhos Pd G2 and heated to about 80° C. under microwaveconditions to give compound 8.

One skilled in the art will recognize that Step D, the Suzuki crosscoupling reaction, could be completed before the azetidine etherformation of Step C.

In Step E, one skilled in the art will recognize that compound 8 may becyclized by the initial reduction of the ketone. This can beaccomplished using a reducing agent, such as lithium triethylborohydride in solvents such as 1,4-dioxane and THF and at a temperatureof about 0° C. to room temperature to give the corresponding secondaryalcohol. This intermediate alcohol can be carried on crude and bedeprotonated with a suitable base such as cesium carbonate, sodiumhydride, sodium t-butoxide or potassium t-butoxide in a solvent such asTHF, DMSO, or DMF. The resulting alkoxide can cyclize into the arylfluoride at room temperature, with heating to reflux, or at atemperature of about 60° C. The substituted cyclic ether formed upondisplacement of the fluoride can then be obtained to give compounds ofFormula I.

Alternatively, the ketone, 8, can be reduced to the alcohol and chirallypurified at Step F to give the chiral alcohol 9, and then cyclized inStep G as described above for Step E to give compounds of Formula I.

In another alternative reaction, the ketone can be reduced using achiral reagent such as (R)-(+)-α.α-diphenyl-2-pyrrolidinemethanol alongwith trimethyl borate and borane-dimethylsulfide to directly give thedesired chiral alcohol, compound 9 which can then be cyclized in Step Gas described above for Step E to give compounds of Formula I.

In an optional step, a pharmaceutically acceptable salt of a compound ofFormula I, Formula II, and Formula III as described herein can be formedby reaction of an appropriate free base of a compound of Formula I,Formula II, and Formula III as described herein with an appropriatepharmaceutically acceptable acid in a suitable solvent under standardconditions. Additionally, the formation of such salts can occursimultaneously upon deprotection of a nitrogen-protecting group. Thepossible formation of pharmaceutically acceptable salts is well known.See, for example, Gould, P. L., “Salt selection for basic drugs,”International Journal of Pharmaceutics, 33: 201-217 (1986); Bastin, R.J., et al. “Salt Selection and Optimization Procedures forPharmaceutical New Chemical Entities,” Organic Process Research andDevelopment, 4: 427-435 (2000); and Berge, S. M., et al.,“Pharmaceutical Salts,” Journal of Pharmaceutical Sciences, 66: 1-19,(1977). One of ordinary skill in the art will appreciate that a compoundof Formula I, Formula II, and Formula III as described herein is readilyconverted to and may be isolated as a pharmaceutically acceptable salt.Examples of useful salts include, but are not limited to,benzenesulfonic acid salts and 4-methybenzenesulfonic acid salts.4-methylbenzenesulfonic acid salts are also known as tosylate salts.

Preparation 1 2-[3-(Fluoromethyl)azetidin-1-yl]ethan-1-ol

Add sodium triacetoxyborohydride (405 g, 1.91 mol) portion-wise over aperiod of 15 minutes to a stirred 0° C. solution of3-(fluoromethyl)azetidine hydrochloride (160 g, 1.28 mol) in DCM (2.4 L)under N₂ and stir at 0° C. for 10 minutes. Add 1,4-dioxane-2,5-diol (99g, 0.83 mol) at 0° C. in 6 portions over a period of 1 hour then stir at0-5° C. for 15 minutes. Allow the reaction to warm to room temperatureand stir for 2 hours under N₂. Cool the reaction to 10-15° C. over aperiod of 20 minutes, then warm to 25-30° C. and maintain at thistemperature for 2 hours. Add water (800 mL) over a period of 25-30minutes at 10-15° C., allow to warm to room temperature for 5-10 minutesand then separate the layers. Wash the aqueous layer with DCM (800 mL),separate the layers then cool the combined aqueous layers to 10-15° C.and adjust the pH to 13-14 using 50% sodium hydroxide solution (˜540mL). Allow the aqueous layer to warm to room temperature, extract withDCM (4×800 mL), dry with sodium sulfate (80 g), filter, and concentrateto dryness to obtain the title compound (139 g, 82%) as a thick yellowoil. ES/MS (m/z): 134.1 (M+H).

Preparation 2 2-[3-(Fluoromethyl)azetidin-1-yl]ethan-1-ol hydrochloride

Dissolve 2-[3-(fluoromethyl)azetidin-1-yl]ethan-1-ol (529 g, 4 mol) inMTBE (2.6 L) and cool to 0° C. Add HCl/EtOH solution (492 mL, 30 wt %)drop-wise over 30 minutes then stir at 0° C. for 30 minutes. Filter thesolids and wash the filter cake with MTBE (2×200 mL). Dry under N₂ for 8hours to obtain the title compound (580 g, 86%) as a white solid. ES/MS(m/z): 134.0 (M+H).

Preparation 3(3-Chloro-7-methoxyquinolin-4-yl)-(4-fluorophenyl)methanone

Cool a mixture of 4-bromo-3-chloro-7-methoxyquinoline (70 g, 254 mmol)and THF (1 L) to −40° C. under N₂ resulting in precipitation of thematerial. Add isopropylmagnesium chloride (2 M in THF, 254 mL, 509 mmol)over 20 minutes and stir the mixture for 1 hour. Add a solution of4-fluorobenzoyl chloride (66 mL, 559 mmol) in THF (140 mL) drop-wisethen allow to warm to room temperature. Quench the reaction withsaturated NH₄Cl solution (300 mL) and water (200 mL) and separate thelayers. Wash the organic layer with saturated NH₄Cl solution (300 mL),dry over MgSO₄, filter, and concentrate to provide an oily residue.Filter the crude brown oil through silica gel eluting with a mixture ofMTBE/hexanes (1:1) to obtain the crude product as a yellow solid (84 g).Treat the solid with 10% methylacetate/heptane (800 mL) and stir at roomtemperature overnight. Filter to collect the solids and reserve.Concentrate the filtrate and purify on silica gel eluting with 10-40%EtOAc/hexanes then treat the product with 10% methylacetate/heptane (200mL) and stir at room temperature for 3 hours. Filter the resultingsolids, combine with solids from the previous filtration and dry undervacuum overnight to obtain the title compound (31 g, 38%) as a yellowsolid. ES/MS (m/z): 316.0 (M+H).

Preparation 4(3-Chloro-7-hydroxyquinolin-4-yl)-(4-fluorophenyl)methanone

Add boron tribromide (1 M in DCM, 295 mL, 295 mmol) to a mixture of(3-chloro-7-methoxyquinolin-4-yl)-(4-fluorophenyl)methanone (31 g, 98mmol) in DCM (217 ml) and stir the mixture at room temperature for 3days. Pour the mixture slowly into a 0° C. solution of dibasic potassiumphosphate (2 M in water, 700 mL) and water (200 mL). Allow the mixtureto warm to room temperature and stir for 1 hour. Concentrate thesolution in vacuo to remove organic solvents, filter, collect thefiltrate and dry the filtrate under vacuum at 45° C. overnight. Treatthe solids with DCM/heptane (1:1, 450 mL) and stir overnight. Collectthe solids and dry under vacuum overnight to obtain the title compound(32 g, quantitative yield) as a light brown solid. ES/MS (m/z): 302.0(M+H).

Preparation 5(3-Chloro-7-hydroxyquinolin-4-yl)-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)methanone

Add 2-[3-(fluoromethyl)azetidin-1-yl]ethan-1-ol hydrochloride (3.90 g,23.0 mmol) to a stirred solution of(3-chloro-7-hydroxyquinolin-4-yl)-(4-fluorophenyl)methanone (5.00 g,15.3 mmol) in DMF (75 ml) followed by sodium hydride (60% in mineraloil, 3.02 g, 76.8 mmol). Stir under N₂ and warm to 40° C. for 45minutes. Quench the solution with water and concentrate. Partition theresidue between 20% iPrOH/CHCl₃ and saturated aqueous sodium bicarbonatesolution and separate, extract the aqueous with 2×20% iPrOH/CHCl₃,combine the organic extracts, dry the combined organic layers overmagnesium sulfate, filter and concentrate the filtrate to obtain thecrude product as a dark red oil. Purify the crude material by silica gelcolumn chromatography eluting with a gradient of 5-10% 7 N NH₃ inMeOH/DCM to give the title compound (5.31 g, 84%) as a yellow solid.ES/MS (m/z): 415.0 (M+H).

Preparation 6(4-{2-[3-(Fluoromethyl)azetidin-1-yl]ethoxy}phenyl){3-[2-fluoro-4-(trifluoromethyl)phenyl]-7-hydroxyquinolin-4-yl}methanone

Degas with N₂ (5×) a mixture(3-chloro-7-hydroxyquinolin-4-yl)-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)methanone(200 mg, 0.48 mmol), 2-fluoro-4-(trifluoromethyl)phenylboronic acid (158mg, 0.72 mmol), potassium carbonate (202 mg, 1.45 mmol),2-methyl-2-butanol (3 ml), and water (1 ml) in a microwave vial. AddXPhos Pd G2 (12 mg, 0.015 mmol), seal and microwave at 80° C. for 2hours. Partition the residue between MTBE and saturated NH₄Cl solution.Separate the layers and extract the aqueous with MTBE. Combine theorganic extracts, dry over magnesium sulfate, filter, and concentratethe filtrate to obtain an orange residue. Purify the crude material bysilica gel column chromatography eluting with 5% MeOH/DCM to give thetitle compound (205 mg, 78%) as a yellow solid. ES/MS (m/z): 543.2(M+H).

Prepare the following compounds in a manner essentially analogous to themethod of Preparation 6, with the following variations in procedure,heating times between 1-2 hours, extraction with MTBE or EtOAc, anddrying of organic layers over magnesium sulfate or sodium sulfate.Purify by silica gel column chromatography using up to 10% (MeOH or 7 Mammoniated MeOH) in DCM (Prep 10: gradient 3-8% 7 M ammoniated MeOH inDCM; Preps 9 and 11: gradient 4 to 10% 7 M ammoniated MeOH in DCM)and/or by high pH reversed phase chromatography as noted.

TABLE 2 Compounds prepared according to Preparation 6 Prep ES/MS (m/z)No. Chemical Name Structure (M + H)  7 (4-{2-[3-(Fluoromethyl)azetidin-1- yl]ethoxy}phenyl) {3-[2-fluoro-3-(trifluoromethyl) phenyl]-7- hydroxyquinolin- 4-yl}methanone

543.0  8^(a) [3-(4-Chloro-2- fluorophenyl)-7- hydroxyquinolin-4-yl](4-{2-[3- (fluoromethyl) azetidin-1- yl]ethoxy}phenyl) methanone

509.0  9^(b) [3-(3-Chloro-2- fluorophenyl)-7- hydroxyquinolin-4-yl](4-{2-[3- (fluoromethyl) azetidin-1- yl]ethoxy}phenyl) methanone

509.0 10 [3-(2,4- Difluorophenyl)-7- hydroxyquinolin- 4-yl](4-{2-[3-(fluoromethyl) azetidin-1- yl]ethoxy}phenyl) methanone

493.0 11 [3-(2,3- Difluorophenyl)-7- hydroxyquinolin- 4-yl](4-{2-[3-(fluoromethyl) azetidin-1- yl]ethoxy}phenyl) methanone

493.0 12 (4-{2-[3- (Fluoromethyl) azetidin-1- yl]ethoxy}phenyl)[3-(2-fluoro-4- methylphenyl)-7- hydroxyquinolin- 4-yl]methanone

489.2 13 (4-{2-[3- (Fluoromethyl) azetidin-1- yl]ethoxy}phenyl)[3-(2-fluoro-3- methylphenyl)-7- hydroxyquinolin- 4-yl]methanone

489.2 ^(a)Purify by high pH reversed phase flash chromatography (RediSepRf GOLD ® High Performance C18 column, eluting with 35-45% ACN in 10 mMaqueous ammonium bicarbonate with 5% MeOH). ^(b)After purification onsilica elute with 4-10% 7M ammoniated MeOH in DCM, further purify byhigh pH reversed phase flash chromatography (RediSep Rf GOLD ® HighPerformance C18 column, eluting with 30-44% ACN in 10 mM aqueousammonium bicarbonate with 5% MeOH).

Preparation 14 Racemic4-{2-[3-(Fluoromethyl)azetidin-1-yl]ethoxy}phenyl)(hydroxy)methyl]-3-[2-fluoro-4-(trifluoromethyl)phenyl]quinolin-7-ol

Add (4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl){3-[2-fluoro-4-(trifluoromethyl)phenyl]-7-hydroxyquinolin-4-yl}methanone(305 g, 562.2 mmol) and THF (1.5 L) together under N₂ and cool thesolution to 0-5° C. Add lithium triethylborohydride (1 M in THF, 1.5 L,1.5 mol) dropwise. Stir the mixture at 0-5° C. for 1 hour. Add water(300 mL) dropwise and saturated NH₄Cl (1 L). Warm the mixture to roomtemperature. Add EtOAc (2 L) and collect the organic layer. Wash theorganic layer with brine (500 mL), dry over MgSO₄, filter, andconcentrate to dryness. Dissolve the residue in 95:5 mixture of acetoneand 2 M ammonia in MeOH and filter through silica gel to give the titlecompound (264 g, 86.2%) as an orange solid. ES/MS (m/z): 545.2 (M+H).

Preparation 154-{2-[3-(Fluoromethyl)azetidin-1-yl]ethoxy}phenyl)(hydroxy)methyl]-3-[2-fluoro-4-(trifluoromethyl)phenyl]quinolin-7-ol,Isomer 1

Purify Racemic4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)(hydroxy)methyl]-3-[2-fluoro-4-(trifluoromethyl)phenyl]quinolin-7-ol(354 g, 0.62 mol) using chiral chromatography under the followingconditions: Column Chiralpak AD-H, 150×50 mm, flow rate 300 g/minute, UV350 nm, mobile phase 35% iPrOH with 0.5% DMEA/CO₂, column temperature40° C. to give the title compound (171.4 g, 48%) of the first elutingisomer. Confirm enantiomeric enrichment of Isomer 1 by chiral analyticalSFC, >98% ee, t_((R))=0.79 minutes, column: 4.6×150 mm Chiralpak AD-H,eluting with a mobile phase of 35% iPrOH with 0.5% DMEA in CO₂, flowrate of 0.6 mL/minute, UV detection of 350 nm.

Alternate Preparation 15

Add trimethyl borate (65 mg, 0.62 mmol) to a solution of(R)-(+)-α.α-diphenyl-2-pyrrolidinemethanol (132 mg, 0.52 mmol) in THF(20 mL). Stir the mixture under N₂ at room temperature for 1 hour. Addborane-dimethyl sulfide (2.0 M in THF, 2.6 mL, 5.2 mmol) followed by(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl){3-[2-fluoro-4-(trifluoromethyl)phenyl]-7-hydroxyquinolin-4-yl}methanone(1.0 g, 1.73 mmol). Heat the reaction overnight at 45° C. Add additionalborane-dimethylsulfide (2.0 M in THF, 2.6 mL, 5.2 mmol) and stir for 5hours at 45° C. Slowly add saturated NH₄Cl solution (25 mL) and isolatethe organic phase. Re-extract the aqueous extract with 20% iPrOH/CHCl₃.Combine the organic extracts, dry over Na₂SO₄, filter, and evaporate togive a borane complex intermediate (1.2 g). Dissolve one third of theborane complex intermediate (0.4 g, 0.6 mmol) in 1,4-dioxane (4 mL) andethanolamine (0.3 mL, 5 mmol) and heat the reaction to 70° C. for 3hours. Quench the reaction with saturated NH₄Cl solution (25 mL) andisolate the organic phase. Re-extract the aqueous extract with 20%iPrOH/CHCl₃ (4×25 mL). Combine the organic extracts, dry over Na₂SO₄,filter, and concentrate to dryness to give the title compound as anorange solid (0.33 g, 0.57 mmol, 100% yield). LC/MS (m/z): [M+H]⁺ 545.Confirm enantiomeric enrichment of Isomer 1 by chiral analytical SFC,96% ee, t_((R))=0.79 minutes, column: 4.6×150 mm Chiralpak AD-H, elutingwith a mobile phase of 35% iPrOH with 0.5% DMEA in CO₂, flow rate of 0.6mL/minute, UV detection of 350 nm.

Example 1 Racemic5-(4-{2[3-(Fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol

Cool a solution of(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl){3-[2-fluoro-4-(trifluoromethyl)phenyl]-7-hydroxyquinolin-4-yl}methanone(5.27 g, 9.71 mmol) in 1,4-dioxane (100 mL) to 5° C. Add lithiumtriethylborohydride (1 M in THF, 30.0 mL, 30.0 mmol). Remove the coolingbath and stir for 1.5 hours at room temperature. Quench the mixture withwater. Add saturated NH₄Cl solution and EtOAc. Separate the layers andextract the aqueous layer with EtOAc. Combine the organic extracts, dryover anhydrous MgSO₄, filter, and concentrate the filtrate. Dissolve thecrude residue in THF (100 mL). Add sodium hydride (60% in mineral oil,1.94 g, 48.5 mmol). Reflux the solution for 1.5 hours. Add additionalsodium hydride (60% in mineral oil, 1.94 g, 48.5 mmol), then reflux foran additional 30 minutes. Cool the solution to room temperature andquench with water. Add EtOAc and saturated NH₄Cl solution. Separate thelayers and extract the aqueous layer with EtOAc. Combine the organicextract, dry over anhydrous MgSO₄, filter, and concentrate the filtrate.Purify the residue by silica gel column chromatography eluting with agradient of 5-7% MeOH in DCM to give the title compound (3.70 g, 72%) asa light yellow foam. ES/MS (m/z): 525.2 (M+H).

Prepare the following compounds in a manner essentially analogous to themethod of Example 1, with the following variations in procedure. For thereduction, use 3 to 5 equivalents of lithium triethylborohydride withreaction times from 30 minutes to one hour and drying of the organiclayers over magnesium sulfate or sodium sulfate. Use the crude residuedirectly or purify by silica gel column chromatography eluting with agradient of 0-5-7.5-10% MeOH in DCM before cyclization. Complete thecyclization by refluxing in THF for up to 16 hours, or in DMF, from 2hours at room temperature for Ex 2, to 2 hours at 85° C. for Ex 8.Extract with DCM or EtOAc and dry organic layers over magnesium sulfateor sodium sulfate. Purify by silica gel column chromatography using upto 10% (MeOH or 7 M ammoniated MeOH) in DCM (Ex 2: gradient 0-10% MeOHin DCM; Ex 5: gradient 4-10% 7 M ammoniated MeOH in DCM; Ex 8: gradient5-7.5% 7 M ammoniated MeOH in DCM) or by high pH reversed phase HPLC asnoted.

TABLE 3 Example Compounds prepared according to Example 1 Ex ES/MS (m/z)No. Chemical Name Structure (M + H) 2 Racemic 5-(4-{2- [3-(fluoromethyl)azetidin-1- yl]ethoxy}phenyl)-7- (trifluoromethyl)-5H-[1]benzopyrano[4,3- c]quinolin-2-ol

525.2  3^(a) Racemic 8-chloro- 5-(4-{2-[3- (fluoromethyl) azetidin-1-yl]ethoxy}phenyl)- 5H-[1]benzopyrano [4,3-c]quinolin-2-ol

491.0  4^(b) Racemic 7-chloro- 5-(4-{2-[3- (fluoromethyl) azetidin-1-yl]ethoxy}phenyl)- 5H-[1]benzopyrano [4,3-c]quinolin-2-ol

491.0  5^(c) Racemic 8-fluoro- 5-(4-{2-[3- (fluoromethyl) azetidin-1-yl]ethoxy}phenyl)- 5H-[1]benzopyrano [4,3-c]quinolin-2-ol

475.0  6^(d) Racemic 7-fluoro- 5-(4-{2-[3- (fluoromethyl) azetidin-1-yl]ethoxy} phenyl)-5H- [1]benzopyrano [4,3-c]quinolin-2-ol

475.0  7^(c) Racemic 5-(4-{2- [3-(fluoromethyl) azetidin-1-yl]ethoxy}phenyl)- 8-methyl-5H- [1]benzopyrano [4,3-c]quinolin-2-ol

471.2 8 Racemic 5-(4-{2- [3-(fluoromethyl) azetidin-1-yl]ethoxy}phenyl)- 7-methyl-5H- [1]benzopyrano [4,3-c]quinolin-2-ol

471.2 ^(a)Purify by high pH reversed phase HPLC (KINETEX ® C18, 5 μm, 30× 250 mm column, eluting with 35-50% ACN in 10 mM aqueous ammoniumbicarbonate with 5% MeOH). ^(b)Purify by high pH reversed phase HPLC(KINETEX ® C18, 5 μm, 30 × 250 mm column, eluting with 35-43% ACN in 10mM aqueous ammonium bicarbonate with 5% MeOH) ^(c)After purification onsilica eluting with 4-10% 7M ammoniated MeOH in DCM, further purify byhigh pH reversed phase HPLC (KINETEX ® C18, 5 μm, 30 × 250 mm column,eluting with 30-44% ACN in 10 mM aqueous ammonium bicarbonate with 5%MeOH). ^(d)Purify by high pH reversed phase HPLC (XBRIDGE ® C18 5 μmOBD, 30 × 75 mm column, eluting with 10-75% ACN in 10 mM aqueousammonium bicarbonate with 5% MeOH). ^(e)Purify by high pH reversed phaseHPLC (XBRIDGE ® C18 5 μm OBD, 30 × 75 mm column, eluting with 10-60% ACNin 10 mM aqueous ammonium bicarbonate with 5% MeOH).

Example 1A5-(4-{2-[3-(Fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 1 and Example 1B5-(4-{2-[3-(Fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 2

Separate the two enantiomers of5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-olby chiral SFC with the following conditions: Column: LUX® Cellulose-1,5×25 cm; eluting with a mobile phase of 30% iPrOH (with 0.5% DMEA) inCO₂; column temperature: 40° C.; flow rate: 300 g/minute; UV detectionwavelength: 270 nm to give Example 1A as the first eluting enantiomer(Isomer 1). ES/MS (m/z): 525.2 (M+H). Confirm enantiomeric enrichment ofIsomer 1 by chiral analytical SFC, >99% ee, t_((R)): 1.30 minutes;column: CHIRALCEL® OD-H, 4.6×150 mm; eluting with a mobile phase of 30%MeOH (0.2% IPA) in CO₂; column temperature: 40° C.; flow rate: 5mL/minute; UV detection wavelength: 225 nm. Isolate the title compoundof Example 1B to give the second eluting enantiomer (Isomer 2). ES/MS(m/z): 525.2 (M+H). Confirm enantiomeric enrichment of Isomer 2 bychiral analytical SFC, 98% ee, t_((R)): 2.03 minutes; column: CHIRALCEL®OD-H, 4.6×150 mm; eluting with a mobile phase of 30% MeOH (0.2% IPA) inCO₂; column temperature: 40° C.; flow rate: 5 mL/minute; UV detectionwavelength: 225 nm.

Alternate Preparation Example 1B Crystalline5-(4-{2-[3-(Fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 2

Stir5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,4-methylbenzenesulfonic acid, Isomer 2 (23.8 g, 0.034 mol) in water (250mL) at 1000 rpm. Add NaOH (76 μL) and stir the solution for 2 hours. AddDCM (600 mL). Separate the mixture, dry the DCM extract with magnesiumsulfate, filter the material through a syringe filter (0.45 μm), andconcentrate to dryness. Allow the material to sit under a N₂ stream overa weekend. Add 1:1 EtOH/water (80 mL) and stir the mixture withsonication. Collect a tan solid by filtration on a nylon membrane togive the title compound (10.47 g, 0.02 mol, 59%).

X-Ray Powder Diffraction (XRD)

The XRPD patterns of crystalline solids are obtained on a Bruker D4Endeavor X-ray powder diffractometer, equipped with a CuKα source and aVantec detector, operating at 35 kV and 50 mA. The sample is scannedbetween 4 and 40 2θ°, with a step size of 0.008 2θ° and a scan rate of0.5 seconds/step, and using 1.0 mm divergence, 6.6 mm fixedanti-scatter, and 11.3 mm detector slits. The dry powder is packed on aquartz sample holder and a smooth surface is obtained using a glassslide. The crystal form diffraction patterns are collected at ambienttemperature and relative humidity. Crystal peak positions are determinedin MDI-Jade after whole pattern shifting based on an internal NIST 675standard with peaks at 8.853 and 26.774 2θ°. It is well known in thecrystallography art that, for any given crystal form, the relativeintensities of the diffraction peaks may vary due to preferredorientation resulting from factors such as crystal morphology and habit.Where the effects of preferred orientation are present, peak intensitiesare altered, but the characteristic peak positions of the polymorph areunchanged. See, e.g. The United States Pharmacopeia #23, NationalFormulary #18, pages 1843-1844, 1995. Furthermore, it is also well knownin the crystallography art that for any given crystal form the angularpeak positions may vary slightly. For example, peak positions can shiftdue to a variation in the temperature at which a sample is analyzed,sample displacement, or the presence or absence of an internal standard.In the present case, a peak position variability of ±0.2 2θ° is presumedto take into account these potential variations without hindering theunequivocal identification of the indicated crystal form. Confirmationof a crystal form may be made based on any unique combination ofdistinguishing peaks.

Characterize a prepared sample of crystalline5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 2 by an XRD pattern using CuKa radiation as having diffractionpeaks (2-theta values) as described in Table 3 below, and in particularhaving peaks at 19.8 in combination with one or more of the peaksselected from the group consisting of 6.8, 16.0, and 22.1; with atolerance for the diffraction angles of 0.2 degrees.

TABLE 4 X-ray Powder Diffraction Peaks of the Crystalline Example 1BRelative Intensity (% of Peak Angle (°2-Theta) +/− 0.2° most intensepeak) 1 6.8 29.40 2 15.3 8.30 3 16.0 20.10 4 17.4 7.60 5 18.1 16.00 619.8 100.00 7 21.1 14.60 8 22.1 28.90 9 24.9 16.40 10 25.4 21.90

Alternate Preparation Example 1B

Dissolve4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)(hydroxy)methyl]-3-[2-fluoro-4-(trifluoromethyl)phenyl]quinolin-7-ol,Isomer 1 (63.05 g, 104.7 mmol) in DMSO (1.3 L) under N₂ at roomtemperature. Add in portions cesium carbonate (108 g, 331 mmol) over 5minutes. Heat the mixture to 60° C. for 15 hours. Cool the mixture toroom temperature and dilute with water (2.1 L) and EtOAc (1.3 L). Stirthe mixture for 5 minutes and separate. Re-extract the aqueous materialwith EtOAc (1.3 L) and stir for 5 minutes. Separate and combine theorganic extracts, wash with brine, water, and EtOAc. Dry the organicextracts with MgSO₄, concentrate, and dry under high vacuum overnight atroom temperature to give the title compound as a brown solid (52.69 g,95.9%). Confirm enantiomeric enrichment of Example 1B by chiralanalytical SFC, 98.1% ee, t_((R)): 2.03 minutes; column: CHIRALCEL®OD-H, 4.6×150 mm; eluting with a mobile phase of 30% MeOH (0.2% IPA) inCO₂; column temperature: 40° C.; flow rate: 5 mL/minute; UV detectionwavelength: 225 nm.

Example 2A5-(4-{2-[3-(Fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-7-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 1 and Example 2B5-(4-{2-[3-(Fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-7-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 2

Separate the two enantiomers of5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-7-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-olby chiral SFC with the following conditions: Column: CHIRALPAK® IC,21×250 cm; eluting with a mobile phase of 30% iPrOH (with 0.2% IPA) inCO₂; column temperature: 40° C.; flow rate: 70 g/minute; UV detectionwavelength: 225 nm to give Example 2A as the first eluting enantiomer(Isomer 1). ES/MS (m/z): 525.1 (M+H). Confirm enantiomeric enrichment ofIsomer 1 by chiral analytical SFC, >99% ee, t_((R)): 1.56 minutes;column: CHIRALPAK® IC, 4.6×150 mm; eluting with a mobile phase of 30%iPrOH (0.2% IPA) in CO₂; column temperature: 40° C.; flow rate: 5mL/minute; UV detection wavelength: 225 nm. Isolate the title compoundof Example 2B to give the second eluting enantiomer (Isomer 2). ES/MS(m/z): 525.2 (M+H). Confirm enantiomeric enrichment of Isomer 2 bychiral analytical SFC, 98% ee, t_((R)): 2.33 minutes; column: CHIRALPAK®IC, 4.6×150 mm; eluting with a mobile phase of 30% iPrOH (0.2% IPA) inCO₂; column temperature: 40° C.; flow rate: 5 mL/minute; UV detectionwavelength: 225 nm.

Example 3A8-Chloro-5-(4-{2[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 1 and Example 3B8-Chloro-5-(4-{2[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 2

Separate the two enantiomers of8-chloro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-olby chiral SFC with the following conditions: Column: CHIRALCEL® OD-H,21×250 cm; eluting with a mobile phase of 35% MeOH (with 0.2% IPA) inCO₂; column temperature: 40° C.; flow rate: 80 g/minute; UV detectionwavelength: 225 nm to give Example 3A as the first eluting enantiomer(Isomer 1). ES/MS (m/z): 491.0 (M+H). Confirm enantiomeric enrichment ofIsomer 1 by chiral analytical SFC, >99% ee, t_((R)): 1.55 minutes;column: CHIRALCEL® OD-H, 4.6×150 mm; eluting with a mobile phase of 35%MeOH (0.2% IPA) in CO₂; column temperature: 40° C.; flow rate: 5mL/minute; UV detection wavelength: 225 nm. Isolate the title compoundof Example 3B to give the second eluting enantiomer (Isomer 2). ES/MS(m/z): 491.0 (M+H). Confirm enantiomeric enrichment of Isomer 2 bychiral analytical SFC, >99% ee, t_((R)): 2.26 minutes; column:CHIRALCEL® OD-H, 4.6×150 mm; eluting with a mobile phase of 35% MeOH(0.2% IPA) in CO₂; column temperature: 40° C.; flow rate: 5 mL/minute;UV detection wavelength: 225 nm.

Example 4A7-Chloro-5-(4-{2[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 1 and Example 4B7-Chloro-5-(4-{2[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 2

Separate the two enantiomers of7-chloro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-olby chiral SFC with the following conditions: Column: CHIRALCEL® OD-H,21×250 cm; eluting with a mobile phase of 35% MeOH (with 0.2% IPA) inCO₂; column temperature: 40° C.; flow rate: 80 g/minute; UV detectionwavelength: 225 nm to give Example 4A as the first eluting enantiomer(Isomer 1). ES/MS (m/z): 491.0 (M+H). Confirm enantiomeric enrichment ofIsomer 1 by chiral analytical SFC, >99% ee, t_((R)): 1.71 minutes;column: CHIRALCEL® OD-H, 4.6×150 mm; eluting with a mobile phase of 35%MeOH (0.2% IPA) in CO₂; column temperature: 40° C.; flow rate: 5mL/minute; UV detection wavelength: 225 nm. Isolate the title compoundof Example 4B to give the second eluting enantiomer (Isomer 2). ES/MS(m/z): 491.0 (M+H). Confirm enantiomeric enrichment of Isomer 2 bychiral analytical SFC, >99% ee, t_((R)): 2.38 minutes; column:CHIRALCEL® OD-H, 4.6×150 mm; eluting with a mobile phase of 35% MeOH(0.2% IPA) in CO₂; column temperature: 40° C.; flow rate: 5 mL/minute;UV detection wavelength: 225 nm.

Example 5A8-Fluoro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 1 and Example 5B8-Fluoro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 2

Separate the two enantiomers of8-fluoro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-olby chiral SFC with the following conditions: Column: CHIRALCEL® OD-H,21×250 cm; eluting with a mobile phase of 30% MeOH (with 0.2% IPA) inCO₂; column temperature: 40° C.; flow rate: 80 g/minute; UV detectionwavelength: 225 nm to give Example 5A as the first eluting enantiomer(Isomer 1). ES/MS (m/z): 475.0 (M+H). Confirm enantiomeric enrichment ofIsomer 1 by chiral analytical SFC, >99% ee, t_((R)): 1.56 minutes;column: CHIRALCEL® OD-H, 4.6×150 mm; eluting with a mobile phase of 30%MeOH (0.2% IPA) in CO₂; column temperature: 40° C.; flow rate: 5mL/minute; UV detection wavelength: 225 nm. Isolate the title compoundof Example 5B to give the second eluting enantiomer (Isomer 2). ES/MS(m/z): 475.0 (M+H). Confirm enantiomeric enrichment of Isomer 2 bychiral analytical SFC, >99% ee, t_((R)): 2.29 minutes; column:CHIRALCEL® OD-H, 4.6×150 mm; eluting with a mobile phase of 30% MeOH(0.2% IPA) in CO₂; column temperature: 40° C.; flow rate: 5 mL/minute;UV detection wavelength: 225 nm.

Example 6A 7-Fluoro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 1 and Example 6B7-Fluoro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 2

Separate the two enantiomers of7-fluoro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-olby chiral SFC with the following conditions: Column: CHIRALCEL® OD-H,21×250 cm; eluting with a mobile phase of 35% MeOH (with 0.2% IPA) inCO₂; column temperature: 40° C.; flow rate: 80 g/minute; UV detectionwavelength: 225 nm to give Example 6A as the first eluting enantiomer(Isomer 1). ES/MS (m/z): 475.0 (M+H). Confirm enantiomeric enrichment ofIsomer 1 by chiral analytical SFC, >99% ee, t_((R)): 1.32 minutes;column: CHIRALCEL® OD-H, 4.6×150 mm; eluting with a mobile phase of 35%MeOH (0.2% IPA) in CO₂; column temperature: 40° C.; flow rate: 5mL/minute; UV detection wavelength: 225 nm. Isolate the title compoundof Example 6B to give the second eluting enantiomer (Isomer 2). ES/MS(m/z): 475.0 (M+H). Confirm enantiomeric enrichment of Isomer 2 bychiral analytical SFC, >99% ee, t_((R)): 1.95 minutes; column:CHIRALCEL® OD-H, 4.6×150 mm; eluting with a mobile phase of 35% MeOH(0.2% IPA) in CO₂; column temperature: 40° C.; flow rate: 5 mL/minute;UV detection wavelength: 225 nm.

Example 8A5-(4-{2-[3-(Fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-7-methyl-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 1 and Example 8B5-(4-{2[3-(Fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-7-methyl-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 2

Separate the two enantiomers of5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-7-methyl-5H-[1]benzopyrano[4,3-c]quinolin-2-olby chiral SFC with the following conditions: Column: CHIRALCEL® OD-H,21×250 cm; eluting with a mobile phase of 30% iPrOH (with 0.2% IPA) inCO₂; column temperature: 40° C.; flow rate: 80 g/minute; UV detectionwavelength: 265 nm to give Example 8A as the first eluting enantiomer(Isomer 1). ES/MS (m/z): 471.2 (M+H). Confirm enantiomeric enrichment ofIsomer 1 by chiral analytical SFC, >99% ee, t_((R)): 1.47 minutes;column: CHIRALCEL® OD-H, 4.6×150 mm; eluting with a mobile phase of 30%iPrOH (0.2% IPA) in CO₂; column temperature: 40° C.; flow rate: 5mL/minute; UV detection wavelength: 225 nm. Isolate the title compoundof Example 8B to give the second eluting enantiomer (Isomer 2). ES/MS(m/z): 471.2 (M+H). Confirm enantiomeric enrichment of Isomer 2 bychiral analytical SFC, >99% ee, t_((R)): 2.05 minutes; column:CHIRALCEL® OD-H, 4.6×150 mm; eluting with a mobile phase of 30% iPrOH(0.2% IPA) in CO₂; column temperature: 40° C.; flow rate: 5 mL/minute;UV detection wavelength: 225 nm.

Example 95-(4-{2-[3-(Fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 2, benzenesulfonic acid

Heat a slurry of5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 2 (Example 1B) (100 mg, 0.19 mmol) in ACN (3 mL) at 50° C. Add asolution of benzenesulfonic acid monohydrate (40 mg, 0.23 mmol) in ACN(1 mL). Heat the clear yellow solution for 10 minutes at 50° C.Discontinue heating, allow the reaction mixture to cool to roomtemperature, and stir the mixture overnight. Add toluene (2 mL) and stirthe reaction mixture 2 hours. Filter the solution, collect the resultingsolid and wash the solid with ACN (1 mL). Dry the solid under vacuum togive the title compound (74 mg, 55%).

Alternate Preparation Example 9

Heat a slurry of5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 2 (Example 1B) (124.1 mg, 0.24 mmol) in MEK (4 mL) at 50° C. Adda solution of benzenesulfonic acid monohydrate (50 mg, 0.28 mmol)dissolved in MEK (1 mL). Discontinue heating, allow the reaction mixtureto cool to room temperature, and stir the mixture over a weekend.Concentrate under a N₂ stream. Add MEK (1 mL) and slurry to give ayellow crystalline solid. Collect the solid, wash with MEK, and dryunder room temperature vacuum to give the title compound (78.8 mg, 48%).

XRD, Example 9

Complete XRD as described for Example 1B. Characterize a prepared sampleof-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 2, benzenesulfonic acid by an XRD pattern using CuKa radiation ashaving diffraction peaks (2-theta values) as described in Table 4 below,and in particular having peaks at 20.5 in combination with one or moreof the peaks selected from the group consisting of 12.3, 22.2, and 23.1;with a tolerance for the diffraction angles of 0.2 degrees.

TABLE 5 X-ray Powder Diffraction Peaks of the Crystalline Example 9Relative Intensity (% Peak Angle (°2-Theta) +/− 0.2° of most intensepeak) 1 7.6 27.10 2 10.6 34.50 3 12.3 42.10 4 12.6 32.30 5 17.7 32.80 619.2 26.70 7 20.5 100.00 8 22.2 45.50 9 23.1 36.30 10 24.2 29.80

Example 10 Crystalline5-(4-{2-[3-(Fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,4-methylbenzenesulfonic acid, Isomer 2

Add together5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,Isomer 2 (Example 1B) (204.2 g, 389 mmol) and EtOAc (5 L) and stir at60° C. followed by the addition of MeOH (200 mL) at 60° C. to give aclear brown solution. Add the title product (11.48 g) to seed thesolution followed by the addition of a pre-mixed solution of4-methylbenzenesulfonic acid; hydrate (81.4 g, 428 mmol) in EtOAc (800mL) to give a yellow suspension. Stir the suspension for 30 minutes at50° C. Concentrate the suspension to ½ volume. Cool the solution at roomtemperature for 1 hour, filter, collect the solid, and wash the solidwith EtOAc. Dry the solid under vacuum at 30° C. over a weekend to givethe title compound (239 g, 343 mmol). To further purify the material,add the title compound (229 g, 328.7 mmol) and 2-propanol (4.6 L)together and heat to 60° C. for 2 hours. Cool to room temperature for 30minutes. Filter the solid and wash with iPrOH (100 mL). Dry the solidunder a stream of N₂ overnight to give the title compound (174.4 g,76.2%). Combine various lots of the title compound prepared essentiallyin the same manner and add heptane (2 L). Stir the suspension for 30minutes, filter the solid, and wash with heptane (300 mL). Dry thecollected solid under a stream of N₂ overnight to give the titlecompound (199.7 g, 99.5%).

XRD, Example 10

Complete the XRD as described for Example 1B. A prepared sample of5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol,4-methylbenzenesulfonic acid, Isomer 2 (Example 10) is characterized byan XRD pattern using CuKa radiation as having diffraction peaks (2-thetavalues) as described in Table 5 below, and in particular having peaks at20.1 in combination with one or more of the peaks selected from thegroup consisting of 12.8, 19.5, and 22.8; with a tolerance for thediffraction angles of 0.2 degrees.

TABLE 6 X-ray Powder Diffraction Peaks of the Crystalline Ex 10 RelativeIntensity (% of most Peak Angle (°2-Theta) +/− 0.2° intense peak) 1 7.625.70 2 12.4 27.90 3 12.8 36.80 4 18.9 26.50 5 19.5 56.90 6 20.1 100.007 20.9 41.50 8 21.8 40.90 9 22.8 39.40 10 25.4 29.70

Biological Assays

The evidence for a relationship between ER expression and certaincancers is well known in the art.

The results of the following assays demonstrate that the compounds ofFormula I, Formula II, and Formula III of the examples are active SERDsand are conceived to be useful in treating cancer.

ERα (Wild Type), ERα (Y537S Mutant) and ERβ Competition Binding Assay

The purpose of the following ER competition binding assays is todetermine the affinity of a test compound against ERα (wild type), ERα(Y537S mutant), and ERI3.

Run the competition binding assay in a buffer containing 50 mM HEPES, pH7.5, 1.5 mM EDTA, 150 mM NaCl, 10% glycerol, 1 mg/mL ovalbumin, and 5 mMDTT, using 0.025 μCi per well ³H-estradiol (118 Ci/mmol, 1 mCi/mL), 7.2ng/well ERα (wild type), or 7.2 ng/well ERα (Y537S mutant) or 7.7ng/well ERβ receptor. Add the test compound at 10 differentconcentrations ranging from 10,000 nM to 0.5 nM, and determinenonspecific binding in the presence of 1 μM of 1713 estradiol. Incubatethe binding reaction (140 μL) for 4 hours at room temperature, and thenadd cold dextran-charcoal buffer (70 μL) (containing per 50 mL of assaybuffer, 0.75 g of charcoal and 0.25 g of dextran) to each reaction. Mixthe plates for 8 minutes on an orbital shaker at 4° C. and thencentrifuge at 3000 rpm at 4° C. for 10 minutes. Transfer an aliquot (120μL) of the mixture to another 96-well, white flat bottom plate (Costar)and add Perkin Elmer Optiphase Supermix scintillation fluid (175 μL) toeach well. Seal the plates and shake vigorously on an orbital shaker.After an incubation of 2.5 hours, read the plates in a Wallac Microbetacounter. Calculate the IC₅₀ using a 4-parameter logistic curve fit andcalculate % inhibition at 10 μM. Convert the IC₅₀ values for thecompound to K_(i) using Cheng-Prusoff equation. The results of thisassay demonstrate Examples 1, 1A, and 1B (and others) bind torecombinant ERα wild type and ERα mutant (Y537S) as shown in Table 7below and Example 1B was also determined to bind to ERβ with a K_(i)(nM) ERβ competition of 0.11±0.07, n=3.

TABLE 7 ERα (wild type), ERα (Y537S mutant) and ERβ competition bindingresults K_(i) (nM) ERα (wild K_(i) (nM) ERα Example # type) (Y537Smutant) 1 0.87 5.80 1A 12.45 ± 9.32, n = 3 57.18 ± 39.13, n = 3 1B  0.31± 0.38, n = 5 2.79 ± 3.00, n = 5 2 2.17 6.78 2A 0.65 7.92 2B 60.4 293.63 2.36 6.69 3A 8.11 27.23 3B 0.59 2.79 4 0.64 12.11 4A 16.78 54.97 4B0.34 2.34 5 2.82 19.47 5A 12.54 81.15 5B 1.30 6.56 6 4.14 15.77 6A 8.5345.99 6B 1.13 5.71 7 1.55 8.55 8 3.20 11.4 8A 9.33 66.94 8B 0.94 5.44

Of the exemplified compounds tested, the K_(i) for ERα wildtype rangedfrom about 0.300 nM to about 65 nM. The K_(i) for ERα Y537S mutantranged from about 2 nM to 300 nM. The results of this assay demonstratethe binding affinity and potency of the exemplified compounds againstERα wild type, mutant (ESR1 Y537S) and ERβ proteins.

ERα Degradation Assay in MCF7 Cells

The purpose of the following ERα degradation assay is to measure thedegradation of ERα by a test compound in an ERα positive breast cancercell line such as MCF7.

Culture MCF7 (purchased from ATCC HTB-22) cells in DMEM mediasupplemented with 10% FBS, 0.01 mg/mL human insulin 1 and 1%penicillin/streptomycin antibiotics and plate in 384-well flat-bottomplates at a density of 4,000 cells per well in phenol red free DMEMmedia (20 μL) containing 10% charcoal stripped FBS. Incubate the cellsovernight in a cell culture incubator (5% CO₂, 95% relative humidity and37° C.) and allow the cells to attach to the plate. The following daydose the cells with the test compound. Use an Echo 555 acousticdispenser to prepare test compound serial dilutions (1:3) in a rangefrom 6 μM to 0.0003 μM. Dose the cells with the addition of 5 μL fromthe serial dilution plate to the cell plate producing a final DMSOconcentration of 0.2% with a final test compound concentration doserange between 2 and 0.0001 μM. For the maximum point, use mediacontaining 0.2% of DMSO and for the minimum point, use fulvestrantdiluted at 2 μM final concentrations in the growth media containing 0.2%DMSO. After dosing with the test compound, incubate the cell plates at37° C. and 5% CO₂ for 24 hours. Fix the cells by adding 14%para-formaldehyde (10 μL) for 30 minutes at room temperature. Wash thecells once with PBS (20 μL) and incubate with PBS (20 μL) containing0.5% (v/v) TWEEN® 20 for 1 hour. Wash the cells with PBS containing0.05% TWEEN® 20 (2×) and block with 3% BSA in PBS containing 0.05%TWEEN® 20 and 0.1% TRITON™ X-100 (20 μL/well) for 1 hour at roomtemperature. Add 1:500 Primary antibody (20 μL) (ERα (Clone SP1)monoclonal rabbit antibody # RM-9101-S, Thermo Scientific) dilution in1% BSA in PBS containing 0.05% TWEEN® 20 per well, seal the plates andincubate overnight at 4° C. The following day wash the cells with PBScontaining 0.05% TWEEN® 20 (2×) and incubate with secondary antibody (20μL/well) (1:1000 dilution, Goat anti-rabbit IgM ALEXA FLUOR™ 488) in PBS1% BSA for 105 minutes at room temperature. After washing plates withPBS (2×20 μL), add RNase (Sigma) (20 μL of 50 μg/mL) and 1:1000propidium iodide dilution in PBS per well (20 μL). Seal the plates andincubate 1 hour at room temperature on the bench (preserved from light).Scan the plates with ACUMEN EXPLORER™ [Laser-scanning fluorescencemicroplate cytometer manufactured by TTP LABTECH LTD] to measure ERa.Image analysis is based on cellular fluorescent signals for identifyingpositive cells. Identify ER positive cells by mean intensity. Use totalintensity at 575-640 nm from propidium iodide/DNA to identify individualcells. Assay output is % ER positive cells. Determine the IC₅₀ by curvefitting to a four parameter logistic for each output using GENE DATA™.The results of this assay demonstrate potent degradation of ERα inducedby the compounds of Formula I, Formula II, and Formula III as describedherein in MCF7 breast cancer cells. The Relative IC₅₀ values forExamples 1, 1A, and 1B are shown in Table 8.

TABLE 8 ERα degradation assay in MCF7 cells Example # Relative IC₅₀ (μM)1 0.003405 ± 0.001086, n = 3 1A 0.3940 ± 0.1941, n = 4 1B  0.003088 ±0.001523, n = 19 2  0.05220 ± 0.006508, n = 2 2A 0.05125 ± 0.01626, n =2 2B >2 3  0.03347 ± 0.007830, n = 3 3A 0.3905 3B 0.008664 4  0.02241 ±0.0003553, n = 3 4A 0.4998 4B 0.006892 5 0.03653 ± 0.03738, n = 2 5A0.5221 5B 0.009493 ± 0.001103, n = 2 6  0.05086 ± 0.006889, n = 3 6A0.1753 6B 0.009132 7  0.07879 ± 0.007379, n = 2 8  0.01738 ± 0.008752, n= 2 8A 0.2341 8B 0.009617 ± 0.005198, n = 2 10  0.004216 ± 0.001619, n =5

Specifically, the results in Table 7 show potent degradation of ERα bythe compound of Example 1 in MCF7 breast cancer cells. Of theexemplified compounds tested, the relative IC₅₀ ranged from 0.003 to >2μM indicating that all but Example 2B showed activity at theconcentration tested. The results of this assay demonstrate that thecompound of Formula (I) is a SERD with potent ERα degradation activityin cells.

PRα Induction Assay in MCF7 Cells

The purpose of the following PRα induction assay is to determine whethera test compound has agonistic activity against ERα receptor (an agonistwould be expected to activate the receptor).

Culture MCF7 (purchased from ATCC HTB-22) in DMEM media supplementedwith 10% FBS, 0.01 mg/mL human insulin 1 and 1% penicillin/streptomycinantibiotics and plate the cells (prior to becoming 70% confluent) in384-well flat-bottom plates at a density of 4,000 cells per well in 20μL volume in DMEM phenol red free media containing 10% FBS (charcoalstripped). Incubate the cells overnight in a cell culture incubator (5%CO₂, 95% relative humidity at 37° C.) and allow the cells to attach tothe plate. The following day, dose the cells with test compound. Use anEcho 555 acoustic dispenser to prepare compound serial dilutions (1:3)in a range from 6 μM to 0.0003 μM. Dose the cells with the addition ofthe test compound (5 μL) from the serial dilution plate to the cellplate producing a final DMSO concentration of 0.2% with a finalconcentration of the test compound dose range between 2 and 0.0001 μM.For the maximum point use media containing 0.2% of DMSO and for theminimum point, use fulvestrant diluted at 2 μM final concentrations inthe growth media containing 0.2% DMSO. After dosing with the testcompound, incubate the cell plates at 37° C. and 5% CO₂ for 24 hours.Fix the cells by adding 14% para-formaldehyde (10 μL) for 30 minutes atroom temperature. Wash cells once with PBS (20 μL) and incubate with PBS(20 μL) containing 0.5% (v/v) TWEEN® 20 for 1 hour. Wash cells twicewith PBS (20 μL) containing 0.05% TWEEN® 20 and block with 3% BSA in PBScontaining 0.05% TWEEN® 20 and 0.1% TRITON™ X-100 (20 μL/well) for 1hour at room temperature. Add 1:500 primary antibody (20 μL) (PRmonoclonal mouse anti-human antibody, clone PgR 636 Dako, M3569)dilution in 1% BSA/PBS with 0.05 TWEEN® 20 per well, seal the plates andincubate overnight at 4° C.

The following day, wash cells with PBS 0.05% TWEEN® 20 (2×20 μL) andincubate with secondary antibody (20 μL/well) (1:1000 dilution, Goatanti-rabbit IgM ALEXA FLUOR™ 488) in PBS 1% BSA for 105 minutes at roomtemperature. After washing with PBS (2×20 μL), add RNase (20 μL of 50μg/mL) (Sigma) and 1:1000 propidium iodide dilution in PBS per well.Seal plates and incubate 1 hour at room temperature on the bench(preserved from light). Scan plates with ACUMEN EXPLORER™[Laser-scanning fluorescence microplate cytometer manufactured by TTPLABTECH LTD] to measure PRα. Image analysis is based on cellularfluorescent signals for identifying positive cells. Identify PR positivecells by mean intensity. Use total intensity at 575-640 nm frompropidium iodide/DNA to identify individual cells. Assay output is % PRpositive cells. Determine the IC₅₀ by curve fitting to a four parameterlogistic for each output using GENE DATA™. The results of this assaydemonstrate no significant agonistic activity of Examples 1, 1A, and 1Bin MCF7 breast cancer cells. For the compounds tested, the RelativeIC₅₀, in this assay are >2 μM. The results of this assay demonstrate nosignificant agonistic activity of the exemplified compounds tested inMCF7 breast cancer cells. These results also demonstrate that theexemplified compounds tested are antagonists of ERα in MCF7 breastcancer cells (i.e., they have SERD activity).

PRα Inhibition (ERα Functional Antagonism) Cell Assay in MCF7-ESR1 Y537N682 CRISPR Cells

The purpose of the following PRα inhibition (ERα functional antagonism)cell assay is to determine the antagonistic activity of a test compoundagainst the Y537N mutant ERα receptor. An antagonist in this assay isexpected to block the function of the ERα receptor. PRα is a downstreamtranscriptional target of ERα and hence an antagonist of ERα is expectedto inhibit the expression of PRα.

Culture MCF7-ESR1 Y537N-682 (generated by CRISPR/Cas9 gene editing ofESR1 gene in MCF7 cells, clone #682) in DMEM media supplemented with 10%FBS and 1% penicillin/streptomycin antibiotics and plate the cells(prior to becoming 70% confluent) in 384-well flat-bottom plates at adensity of 4,000 cells per well in DMEM phenol red free media 10% FBS(20 μL volume) (charcoal stripped). Incubate the cells overnight in acell culture incubator (5% CO₂, 95% relative humidity and 37° C.) andallow the cells to attach to the plate. The following day dose the cellswith the test compound. Use an Echo 555 acoustic dispenser to preparecompound serial dilutions (1:3) in a range from 6 μM to 0.0003 μM. Dosethe cells with the addition of 5 μL from the serial dilution plate tothe cell plate producing a final DMSO concentration of 0.2% with a finaltest compound concentration dose range between 2 and 0.0001 μM. For themaximum point use media containing 0.2% of DMSO and for the minimumpoint, use fulvestrant diluted at 2 μM final concentrations in thegrowth media containing 0.2% DMSO. After dosing with test compound,incubate the cell plates at 37° C. and 5% CO₂ for 72 hours. Fix thecells by adding 14% para-formaldehyde (10 μL) for 30 minutes at roomtemperature. Wash the cells with PBS (1×20 μL) and incubate with PBS (20μL) of containing 0.5% (v/v) TWEEN® 20 for 1 hour. Wash the cells withPBS (2×20 μL) 0.05% TWEEN® 20, and block with 3% BSA/PBS 0.05% TWEEN®20, 0.1% TRITON™ X-100 (20 μL/well) for 1 hour at room temperature. Add1:500 primary antibody (20 μL) (PR monoclonal mouse anti-human antibody,clone PgR 636 Dako, M3569) dilution in 1% BSA/PBS 0.05 TWEEN® 20 perwell, seal the plates and incubate overnight at 4° C.

The following day, wash the cells with PBS 0.05%® (2×20 μL) and incubatewith secondary antibody (20 μL/well) (1:1000 dilution, Goat anti-rabbitIgM ALEXA FLUOR™ 488) in PBS 1% BSA for 105 minutes at room temperature.After washing with PBS (2×20 μL), add RNase (20 μL of 50 μg/mL) (Sigma)and 1:1000 propidium iodide dilution in PBS per well. Seal the platesand incubate 1 hour at room temperature on the bench (preserved fromlight). Scan the plates with ACUMEN EXPLORER™ [Laser-scanningfluorescence microplate cytometer manufactured by TTP LABTECH LTD] tomeasure PRα. Image analysis is based on cellular fluorescent signals foridentifying positive cells. Identify PR positive cells by meanintensity. Use total intensity at 575-640 nm from propidium iodide/DNAto identify individual cells. Assay output is % PR positive cells.Determine the IC₅₀ by curve fitting to a four parameter logistic foreach output using GENE DATA™.

The results of this assay demonstrate potent inhibition of PRα andfunctional antagonism by Examples 1, 1A, and 1B in MCF7 (ESR1 Y537N,heterozygous mutant) breast cancer cells. The Relative IC_(50s) ofExamples 1, 1A, and 1B (and others) in this assay are shown in Table 9below. The Relative IC_(50s) of the exemplified compounds tested rangefrom about 0.0118 to >1.6 μM indicating the exemplified compounds arepotent antagonist of ERα mutant (Y537N) and potent inhibitors of ERαmediated transcription except example 2B 1.6 PRα (PGR) is also atranscriptional target of ERα and the results from this assaydemonstrate potent inhibition of ERα-mediated transcription of PRα.

TABLE 9 PRα inhibition (ERα functional antagonism) cell assay in MCF7Y537N 682 CRISPR cells Example # Relative IC₅₀ (μM) 1 0.01679 ± 0.00003,n = 2 1A 1.20 ± 0.29, n = 2 1B  0.0130 ± 0.0059, n = 14 2  0.01451 ±0.002619, n = 2 2A  0.02494 ± 0.007386, n = 3 2B  1.639 ± 0.2228, n = 33 0.07717 ± 0.01154, n = 2 3A 0.6117 3B 0.016 4  0.03854 ± 0.003865, n =2 4A 0.5052 4B 0.01181 5 0.06614 ± 0.01551, n = 2 5A 0.3945 5B  0.01822± 0.009815, n = 2 6 0.06319 ± 0.01609, n = 2 6A 0.2364 6B 0.0136 70.1271 8  0.04124 ± 0.006572, n = 2 8A 0.4335 ± 0.1946, n = 3 8B0.008926 ± 0.003828, n = 3 10  0.007936 ± 0.003163, n = 3

PRα Inhibition (ERα Functional Antagonism) Cell Assay in MCF7 Cells

The purpose of the following PRα inhibition (ERα functional antagonism)cell assay is to determine the antagonistic activity of a test compoundagainst the ERα receptor. An antagonist in this assay is expected toblock the function of the ERα receptor. PRα is a downstreamtranscriptional target of ERα and hence an antagonist of ERα is expectedto inhibit the expression of PRα.

Carry out the assay conditions as detailed in the ERα degradation Cellbase Acumen assay above, using the MCF7 cell line except that, prior totest compound dispensing, remove the media from the cell plate andpretreat all wells except for the negative control wells (column 24 ofthe plate) with assay media containing 0.47 nM estradiol for 30 minutes.In this assay, carry out immunostaining for the detection of PRα andscan the plates with ACUMEN EXPLORER™ [Laser-scanning fluorescencemicroplate cytometer manufactured by TTP LABTECH LTD] to measure PRα.Image analysis is based on cellular fluorescent signals for identifyingpositive cells. Identify PRα positive cells by mean intensity. Use totalintensity at 575-640 from propidium iodide/DNA to identify individualcells. Assay output is % PRα positive cells. Determine the IC₅₀ by curvefitting to a four parameter logistic for each output using GENE DATA™.The results of this assay demonstrate potent inhibition of PRα andfunctional antagonism by Examples 1, 1A, and 1B in MCF7 breast cancercells. The Relative IC₅₀ of Examples 1, 1A, and 1B in this assay areshown in Table 10 below. The Relative IC₅₀ of the exemplified compoundsrange from about 0.029 to >2 μM indicating that all exemplifiedcompounds tested except 1A and 2B, are potent antagonists of ERαwild-type protein and a potent inhibitor of ERα mediated transcription.PRα (PGR) is also a transcriptional target of ERα and the results fromthis assay demonstrate potent inhibition of ERα-mediated transcriptionof PRα at the concentration tested.

TABLE 10 PRα inhibition (ERα functional antagonism) cell assay in MCF7cells Example # Relative IC₅₀ (μM) 1 0.1283 ± 0.0226, n = 3 1A >2.000 1B 0.04129 ± 0.03370, n = 16 2 0.1634 2A  0.1215 ± 0.05368, n = 22B >2.000 3 0.07666 ± 0.02101, n = 3 3A 0.9274 3B 0.03435 4 0.07626 ±0.1676, n = 3  4A 0.8465 4B 0.02866 5  0.1180 ± 0.01230, n = 2 5A 0.60025B  0.03203 ± 0.005306, n = 2 6  0.08258 ± 0.005682, n = 3 6A 0.2528 6B0.02835 7  0.1134 ± 0.02087, n = 2 8 0.06835 ± 0.02273, n = 2 8A 0.20588B 0.04848 ± 0.02944, n = 2 10   0.02633 ± 0.004459, n = 3

Cell Proliferation Assay in MCF7 and MCF7-ESR1 Y537N-682

The purpose of the following cell proliferation assays generally is todetect whether a test compound has effects on cell proliferation.

Seed MCF7 (purchased from ATCC HTB-22) cells at a density of 2,000 cellsper well in DMEM phenol red free media 10% FBS (20 μL volume) (charcoalstripped) into a clear bottom 384-well cell culture plate. PlateMCF7-ESRY537N-682 (generated by CRISPR/Cas9 gene editing of ESr1 gene inMCF7 cells, clone #682) in DMEM media supplemented with 10% FBS, and 1%penicillin/streptomycin antibiotics at a density of 1000 cells per well.Incubate the plates at 37° C. and 5% CO₂. The following day dose thecells with the test compound. Use an Echo 555 acoustic dispenser toprepare test compound serial dilutions (1:3) in a range from 60 μM to0.003 μM. Dose the cells with the addition of 5 μL from the serialdilution plate to the cell plate, producing a final DMSO concentrationof 0.2% with a final test compound concentration dose range between 20and 0.001 μM. For the maximum point use media containing 0.2% of DMSOand for the minimum point use fulvestrant diluted at 2 μM finalconcentrations in the growth media containing 0.2% DMSO. After dosingwith the test compound, incubate the cell plates at 37° C. and 5% CO₂.Seven days after test compound addition, remove the plates from theincubator and add cold EtOH 96% (65 μL) to each well. After 30 minutes,remove the media and add RNase (20 μL of 50 μg/mL) (Sigma) and 1:1000propidium iodide dilution in PBS per well. Seal the plates and incubate1 hour at room temperature on the bench (preserved from light). Scan theplates with ACUMEN EXPLORER™ [Laser-scanning fluorescence microplatecytometer manufactured by TTP LABTECH LTD]. The MCF-7 cell line growsforming aggregates, cell number as number of objects may not be able tobe used as readout; so the cell number may be evaluated throughestimated number of cells (calculated through the area parameter (ratioof total area of the total cells population (a designated range of peakintensity of FL-1 (PI) and the mean area of the single cells population(defined by perimeter)). Determine the IC₅₀ by curve fitting to a fourparameter logistic for each output using GENE DATA™. The Relative IC₅₀of Examples 1, 1A, and 1B (and others) in MCF7 ESR1 wild type andMCF7-ESR1 Y537N mutant cells are shown in Table 10 below. The results ofthis assay demonstrate potent anti-proliferative activity and cellgrowth inhibition by Examples 1, 1A, and 1B (and others) in MCF7 (ESR1wild type) and MCF7 (ESR1 Y537N mutant) breast cancer cells. TheRelative IC₅₀ of the exemplified compounds range from about 0.0035 to1.176 μM in MCF7 ESR1 wild type and 0.014 to 1.86 μM in MCF7 (ESR1 Y537Nmutant) breast cancer cells indicating that all exemplified compoundstested demonstrate potent anti-proliferative activity and cell growthinhibition in MCF7 (ESR1 wild type) and MCF7 (ESR1 Y537N mutant) breastcancer cells.

TABLE 11 Cell Proliferation Assay in MCF7 and MCF7-ESR1Y537N-682Relative IC₅₀ (μM) Relative IC₅₀ (μM) MCF7 ESR1 MCF7 ESR1 Y537N Example# wild type mutant cells 1 0.00768 ± 0.01263, n = 3  0.0321 ± 0.0068, n= 2  1A 1.18 ± 0.68, n = 4  1.86 ± 1.03, n = 4  1B 0.00349 ± 0.00225, n= 11 0.0167 ± 0.0091, n = 12 2 0.00425 0.05113 2A 0.00612 0.04284 ±0.002666, n = 2 2B 0.4053 0.7777 3 0.3287 0.02394 3A 0.303 0.6169 ±0.1735, n = 3  3B 0.008785 0.02144 ± 0.008938, n = 3 4 0.02861 0.026644A 0.2862 0.5442 ± 0.2181, n = 3  4B 0.003496 0.01433 ± 0.004925, n = 35 0.08009 0.07252 ± 0.02632, n = 2  5A 0.4095 0.5167 ± 0.09497, n = 3 5B0.007666 0.02131 ± 0.01300, n = 3  6 0.05128 0.02362 6A 0.0759 0.3234 ±0.1758, n = 3  6B 0.01539 7 0.01902 0.04479 ± 0.01188, n = 2  8 0.041570.03290 ± 0.003002, n = 2 8A 0.1743 0.6621 ± 0.1173, n = 2  8B 0.0050830.01419 ± 0.01108, n = 2  10  0.004379 0.01059

In Vivo Target Inhibition (IVTI) Assay (PGR RT-qPCR Assay) in MCF7Tumors

The purpose of this IVTI assay is to measure the ability of a testcompound (SERD) to inhibit PRα gene expression (transcription)downstream of ERα in xenograft tumors implanted in mice.

Implant female NOD SCID mice (22-25 g) from Envigo RMS, Inc., Madison,Wis. with 5×10e⁶ MCF7 ER-positive breast cancer cells (ATCC, # HTB-22)subcutaneously in the right flank region in 1:1 HBSS+MATRIGEL™ solution(200 μL). Implant a 17-β estradiol pellet (0.18 mg/pellet, 90 dayrelease, from Innovative research) subcutaneously 1 day prior to tumorcell implantation. Measure tumor growth and body weight twice per weekbeginning the seventh day after the implantation. When tumor sizes reach250-350 mm³, randomize animals and group into groups of five animals.Dose animals with either the test compound at multiple doses in a testcompound specific vehicle (1% hydroxyethylcellulose/0.25% TWEEN®80/0.05% Antifoam in purified water) or vehicle alone orally for 3 daysand collect tumors and blood at desired time intervals after last dose.Sacrifice animals using isoflurane anesthesia plus cervical dislocation.Flash freeze tumors and store at −80° C. until processing for RNAisolation and RT-qPCR assay. Collect blood in EDTA tubes, spin down forplasma, and freeze at −80° C. in a 96-well plate. Determine testcompound exposures using mass spectrometry.

Pulverize tumors in liquid nitrogen and lyse in 1 xRNA lysis buffer(from RNA isolation kits) using Matrix D beads (MP Biomedical,#6913-500) in a FASTPREP-24™ Cell Disrupter machine (MP Biomedical).Transfer tumor lysates to fresh tubes after spinning at 14000 rpm for 20minutes at 4° C. Isolate RNA from tumor lysates using PURELINK® RNA MiniKit (Invitrogen #12183018A) or RNeasy Mini Kit (Qiagen #74104 and#74106). Remove DNA contaminants using PURELINK® DNase Set (Invitrogen#12185010) or RNase-Free DNase Set (Qiagen #79254). Measure isolated RNAconcentration by diluting samples in RNase free water and measuring theabsorbance at 260 nm on a plate reader (SpectraMax190). Subtract theaverage 260 nm absorbance measurement of the blank (RNase free wateronly) from the 260 nm measurements of all other RNA samples. Dilute RNAsamples to equal concentrations in RNase free water. Synthesize cDNAfrom diluted RNA using First-Strand Synthesis System for RT-PCR(Invitrogen, #18080-051). To perform RT-qPCR, first dilute cDNA in RNasefree water. Combine 2× Absolute Blue qPCR ROX Mix (Thermo, # AB-4139/A),PGR primer (Thermo, Hs01556702_m1), and diluted cDNA for each reactionin a PCR plate (Applied Biosystems, #4309849). Amplify cDNA byincubating the samples for 2 minutes at 50° C. followed by 15 minutes at95° C. in the thermocycler (ABI Prism 7900HT Sequence Detection System).Continue to incubate at 95° C. for 15 seconds followed by 50° C. for 60seconds for a total of 40 cycles. Cycles are normalized to thehousekeeping gene and used to calculate % PGR inhibition compared to thevehicle alone. Analyze each sample in duplicate and use average numbersfor calculations. Calculate the percent target (PGR) inhibition usingExcel and XL Fit.

The results of this assay demonstrates that Example 1B inhibits PRα(PGR) expression in the tumor xenograft model. Example 1B inhibits PRα(PGR) expression by ˜78% in the tumor xenograft model for 24 hours with30 mg/kg dose when administered orally. These results demonstratesignificant and sustained inhibition of ERα antagonistic activity andERα-mediated transcriptional activity in vivo in a tumor xenograftmodel.

In Vivo Tumor Growth Inhibition Study in ER-Positive (ESR1 Wild Type)Breast Cancer Xenograft Tumor Models Implanted in Mice

The purpose of the following xenograft tumor inhibition assay is tomeasure reduction in tumor volume in response to test compoundadministration.

Expand human breast cancer cells MCF7 (ATCC # HTB-22) and HCC1428 (ATCC# CRL-2327) in culture, harvest and inject 5×10e⁶ cells in 1:1HBSS+MATRIGEL™ solution (200 μL) subcutaneously on to the rear rightflank of female NOD SCID mice (22-25 g, Envigo RMS, Inc). Twenty-fourhours prior to implantation of cells, implant estrogen pellets (0.18mg/pellet, 17β estradiol, 90-day release, Innovative Research)subcutaneously. Expand human breast cancer cells T47D (ATCC # HTB-22) inculture, harvest and inject 5×10e⁶ cells in 1:1 HBSS+MATRIGEL™ solution(200 μL) subcutaneously on to the rear right flank of female NOD SCIDmice (22-25 g, Envigo RMS, Inc). Twenty-four hours prior to implantationof cells, implant estrogen pellets (0.38 mg/pellet, 17β estradiol,90-day release, Innovative Research) subcutaneously. Expand human breastcancer cells ZR-75-1 (ATCC # CRL-1500) in culture, harvest and inject5×10e⁶ cells in 1:1 HBSS+MATRIGEL™ solution (200 μL) subcutaneously onto the rear right flank of female NOD SCID mice (22-25 g, Envigo RMS,Inc). Twenty-four hours prior to implantation of cells, animals areadministered with 50 μl of estradiol valerate (Delestrogen®)intramascular injection (10 mg/mL) and then once every 14 days for theduration of the study. Measure tumor growth and body weight twice perweek beginning the seventh day after the implantation. When tumor sizesreach 250-350 mm³, randomize animals and group into groups of 5 animals.Prepare the test compound, Example 1B in an appropriate vehicle (1%hydroxyethylcellulose/0.25% TWEEN® 80/0.05% Antifoam in purified water)and administer by oral gavage for 28 days, QD. Determine tumor responseby tumor volume measurement performed twice a week during the course oftreatment. Take the body weight as a general measure of toxicitywhenever tumor volume is measured.

The compound of Example 1B is found to have delta T/C % values asprovided in Table 12 below. These results indicate that the compound ofExample 1B demonstrates good oral bioavailability in mice andsignificant anti-tumor activity or tumor regressions in ER-positive(ESR1 wild-type) human breast cancer xenograft models.

TABLE 12 In vivo tumor growth inhibition study in ER-positive breastcancer xenograft tumor models implanted in mice Delta T/C % or TumorModel Dose (mg/kg) Regression % p-value MCF7 (Breast 3 −26 0.001* CancerXenograft) 10 −46 <0.001* 30 −36 <0.001* T47D (Breast 3 30 0.008* CancerXenograft) 10 −11 <0.001* 30 −28 <0.001* ZR-75-1 (Breast 3 4 <0.001*Cancer Xenograft) 10 0 <0.001* 30 19 <0.001* HCC1428 (Breast 10 −45<0.001* Cancer Xenograft) 30 −22 <0.001* Analysis for tumor volume isbased on Log 10 and SpatialPower covariance structure. *significant (p <0.05) compared to vehicle control. Delta T/C % is calculated when theendpoint tumor volume in a treated group is at or above baseline tumorvolume. The formula is 100*(T − T₀)/(C − C₀), where T and C are meanendpoint tumor volumes in the treated or control group, respectively. T₀and C₀ are mean baseline tumor volumes in those groups. Regression % iscalculated when the endpoint volume is below baseline. The formula is100*(T − T₀)/T₀, where T₀ is the mean baseline tumor volume for thetreated group.Grand mean of all groups from baseline (randomization) at day 32 is usedto compute % change of T/C.

In Vivo Tumor Growth Inhibition Study in ESR1 Mutant (Y537S) BreastCancer PDX Tumor Model (ST941/HI) Implanted in Mice

The purpose of the following xenograft tumor inhibition assay is tomeasure reduction in tumor volume in response to test compoundadministration in an ESR1 mutant and hormone-independent (HI) breastcancer patient-derived xenograft (PDX) model.

ST941/HI PDX model was derived at and run at South Texas AcceleratedResearch Therapeutics (San Antonio, Tex.). Tumor fragments wereharvested from host animals and implanted into immune-deficient mice(The Jackson Laboratory) and the study was initiated at a mean tumorvolume of approximately 125-250 mm³. Prepare the test compound, Example1B in an appropriate vehicle (1% hydroxyethylcellulose/0.25% TWEEN®80/0.05% Antifoam in purified water) and administer by oral gavage for28 days. Determine tumor response by tumor volume measurement performedtwice a week during the course of treatment. Take the body weight as ageneral measure of toxicity whenever tumor volume is measured.

The compound of Example 1B is found to have delta T/C % values asprovided in Table 13 below. These results indicate that the compound ofExample 1B demonstrates good oral bioavailability in mice andsignificant anti-tumor activity or tumor regressions in an ESR1 mutant(Y537S) human breast cancer PDX model.

TABLE 13 In vivo tumor growth inhibition study in ESR1 mutant breastcancer PDX tumor model implanted in mice Delta T/C % or Tumor Model Dose(mg/kg) Schedule Regression % p-value ST941C/HI 3 QD 66 0.213 (ESR1Mutant 10 QD 15 <0.001* Breast Cancer 30 QD 6 <0.001* PDX model)Analysis for tumor volume is based on Log 10 and SpatialPower covariancestructure. *significant (p < 0.05) compared to vehicle control. DeltaT/C % is calculated when the endpoint tumor volume in a treated group isat or above baseline tumor volume. The formula is 100 * (T − T₀)/(C −C₀), where T and C are mean endpoint tumor volumes in the treated orcontrol group, respectively. T₀ and C₀ are mean baseline tumor volumesin those groups. Regression % is calculated when the endpoint volume isbelow baseline. The formula is 100 * (T − T₀)/T₀, where T₀ is the meanbaseline tumor volume for the treated group.Grand mean of all groups from baseline (randomization) at day 32 is usedto compute % change of T/C.

Combination Studies

Due to tumor heterogeneity and acquired resistance to endocrinetherapies, combination therapy has become essential in ER-positive andadvanced/metastatic breast cancer treatment for effective therapy or toovercome acquired resistance. We have tested the combination effect ofExample 1B with CDK4/6 inhibitor abemaciclib, mTOR inhibitor everolimus,PIK3CA inhibitor alpelisib and PI3K/mTOR inhibitor8-[5-(1-hydroxy-1-methylethyl)pyridin-3-yl]-1-[(2S)-2-methoxypropyl]-3-methyl-1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one(“Compound A”) in five ER-positive breast cancer cell lines in vitro.

Cell Viability Assay for Combination Studies

Seed cell at the density shown in Table 14 below in 20 μL volume of themedia described in the table into a clear bottom 384-well cell cultureplate.

TABLE 14 Cell viability assay cell line information Fixation SeedingCommercial time Cell Line density reference Cuture Medium (hours) T-47D1000 ATCC HTB-133 RMPI 10% FBS 1% 72 P/S, 0.008 mg/mL Bovine InsulinMCF-7 1000 ATCC HTB-22 DMEM 10% FBS 1% 96 P/S 0.01 mg/mL human insulinEFM-19 3000 ACC 231 RMPI 10% FBS 1% 144 P/S ZR-75-1 1000 ATCC CRL-1500RMPI 10% FBS 1% 144 P/S BT-474 1000 ATCC HTB-20 HYBRI-CARE (1 L 144 H₂O,1.5 g/L sodium bicarbonate, 10% FBS, 1% P/S) ZR-75-30 1000 ATCC CRL-1504RMPI 10% FBS 1% 240 P/S

Incubate the plates at 37° C. and 5% CO₂. The following day dose thecells with the test compound, Example 1B.

Prepare compounds as 10 mM DMSO stock solutions and use for a doseresponse study with top concentration starting at 10 or 1 μM, twocompounds tested together at a fixed ratio, and then 1:3 serialdilutions serial dilution prepared as well as compounds alone for IC₅₀determination with a starting concentration of 20 μM. Dose the cellswith the addition of 5 μL from the serial dilution plate to the cellplate, producing a final DMSO concentration of 0.2% with a final testcompound concentration dose range between 20 and 0.001 μM for singletreatment or lower range for the combinations. For the maximum point usemedia containing 0.2% of DMSO and for the minimum point usestaurosporine diluted at 2 μM final concentrations in the growth mediacontaining 0.2% DMSO. After dosing with the test compound, incubate thecell plates at 37° C. and 5% CO₂. After two doubling times incubationwith the compounds, remove the plates from the incubator and add coldEtOH 96% (65 μL) to each well. After 30 minutes, remove the media andadd RNase (20 μL of 50 μg/mL) (Sigma) and 1:1000 propidium iodidedilution in PBS per well. Seal the plates and incubate 1 hour at roomtemperature (preserved from light). Scan the plates with ACUMENEXPLORER™ [Laser-scanning fluorescence microplate cytometer manufacturedby TTP LABTECH LTD]. As some cell lines grows forming aggregates, cellnumber as number of objects may not be able to be used as readout; sototal area population (a designated range of peak intensity of FL-1(PI)) or total intensity of PI are used to evaluate cell number.

In vitro combination data suggests synergy (as defined below) withcombination of Example 1B with abemaciclib, or everolimus in 5 out of 5ER-positive breast cancer cell lines as shown in Table 15. Combinationof Example 1B with Compound A synergistic in 4 out of 4 ER-positivebreast cancer cell lines tested. Combination effect of Example 1B withalpelisib is additive in 2 out of 4 ER-positive breast cancer cell linesand synergistic in 2 out of 4 ER-positive breast cancer cell lines.

TABLE 15 In vitro combination of Example 1B with other targeted agentsin ER-positive breast cancer cell lines Breast cancer cell CombinationStatistical Biological line Treatment 1 Treatment 2 Index (CI50)Interpretation Interpretation MCF7 Example 1B Abemaciclib 0.2675Synergistic Additive Everolimus 0.0389 Synergistic Synergistic T47DExample 1B Abemaciclib 0.2693 Synergistic Synergistic Everolimus 0.0609Synergistic Synergistic alpelisib 0.0818 Synergistic SynergisticCompound A 0.2401 Synergistic Synergistic ZR-75-1 Example 1B Abemaciclib0.2067 Synergistic Synergistic Everolimus 0.1853 Synergistic Synergisticalpelisib 1.3717 Additive Additive Compound A 0.3768 SynergisticSynergistic ZR-75-30 Example 1B Abemaciclib 0.3960 SynergisticSynergistic Everolimus 0.1248 Synergistic Synergistic alpelisib 0.4149Additive Additive Compound A 0.7098 Synergistic Synergistic EFM-19Example 1B Abemaciclib 0.3455 Synergistic Synergistic Everolimus 0.5033Synergistic Additive alpelisib 0.2963 Synergistic Synergistic Compound A0.4326 Synergistic Synergistic

Data Analysis and Interpretation of Combination Effect

Use methods that are published in the literature to calculate in vitrocombination effect (L. Zhao, et al, Front Biosci, 2010, 2:241-249 and L.Zhao, et al, Clin Cancer Res, 2004, 10(23):7994-8004). In order toidentify synergistic or antagonistic interactions between two drugs, acurve shift analysis has been performed using a customized XL templatewith XLFit 5 add ins. Single agent's curves are adjusted using 4parameters logistic regression. Criteria and restrictions used for thefitting are (i) bottoms <(−20) are fixed to 0 and (ii) top >120 fixed to100. If all the observations are lower than a threshold set by the user,then a constant fit with hill=0 is performed and the IC₅₀ is consideredhigher than the maximum included concentration. Once the absolute IC₅₀of each single agent has been obtained, the equivalent concentration at50% of activity is calculated for the singles and combination. Usingthese equivalent concentrations together with the measured activities werecalculate an absolute IC₅₀, the curve for single agents will reach 50%activity at values of eq concentrations equals to 1, while synergisticcombinations will reach the 50% at lower values resulting in a leftwardshift, and antagonistic combination will show rightward shift.Equivalent concentrations are also used to calculate CI₅₀ (CombinationIndex at 50% of activity), where CI₅₀ equals absolute IC₅₀ ofcombination curve. Together with CI₅₀ other CI's (Combination Indexes)at different percentages of activity can be calculated (CI10, CI20,CI30, CI40, CI60, CI70, CI80. CI90). In order to calculate Clnn,equivalent concentration at different activity percentage arecalculated. For each activity percentage we calculate the margin oferror which is the confidence interval at 95% and using this confidenceinterval we will calculate upper limit as the addition of margin oferror to the CI and the lower limit as the subtraction of the margin oferror to the CI. Upper limit=CI+Confidence interval 95% and LowerLimit=CI-Confidence interval 95%. These limits are then used tointerpret the results.

Statistical Interpretation at each activity percentage is as follows:

Lower limit < 1 and upper limit > 1 Additive Upper limit < 1 SynergyLower limit > 1 Antagonist

Biological Interpretation at each activity percentage is as follows:

CInn < 0.5 Synergy CInn > 0.5 and CInn < 2 Additive CInn > 2 Antagonist

In Vivo Combination Studies

Due to tumor heterogeneity and acquired resistance to endocrinetherapies, combination therapy has become essential in ER-positive andadvanced/metastatic breast cancer treatment for effective therapy or toovercome acquired resistance. It is hypothesized that a combination oftargeted therapies has the potential to be more effective in slowing oreven halting ER-positive breast cancers. Combination of CDK4/6inhibitors and fulvestrant has been approved for the treatment ofER-positive metastatic breast cancer but a high percentage of patientsdevelop resistance due to acquired mutations in ESR1 or PIK3CA. As apotent degrader and antagonist of ERα, oral SERD such as Example 1B hasthe potential to be more effective in slowing or halting ESR1 mutant orPIK3CA mutant breast cancers as a single agent or in combination withCDK4/6 inhibitor such as abemaciclib or PI3K/mTOR inhibitor such asCompound A. In that context, the compound of Example 1B is tested fortumor growth inhibition in combination with abemaciclib (patentreference) or Compound A (patent reference). More specifically thecompound of Example 1B is tested in combination with abemaciclib orCompound A in ESR1 wild type and PIK3Ca mutant MCF7 breast cancerxenograft model.

Expand human breast cancer cells MCF7 (ATCC # HTB-22) in culture,harvest and inject 5×10e⁶ cells in 1:1 HBSS+MATRIGEL™ solution (200 μL)subcutaneously on to the rear right flank of female NOD SCID mice (22-25g, Envigo RMS, Inc). Twenty-four hours prior to implantation of cells,implant estrogen pellets (0.18 mg/pellet, 17β estradiol, 90-day release,Innovative Research) subcutaneously. Measure tumor growth and bodyweight twice per week beginning the seventh day after the implantation.When tumor sizes reach 250-350 mm³, randomize animals and group intogroups of 5 animals. Prepare the test compound Example 1B in anappropriate vehicle (1% hydroxyethylcellulose/0.25% TWEEN® 80/0.05%Antifoam in purified water) and administer by oral gavage for 42 days.The CDK4/6 inhibitor (abemaciclib) is formulated in 1% HEC in 25 mMsodium phosphate buffer, pH 2.0. The PI3K/mTOR inhibitor (Compound A)was formulated in 1% hydroxyethylcellulose/0.25% TWEEN® 80/0.05%Antifoam in purified water. Determine tumor response by tumor volumemeasurement performed twice a week during the course of treatment. Takethe body weight as a general measure of toxicity whenever tumor volumeis measured. Tumor volume is estimated by using the formula v=1×w2×0.535where 1=larger of measured diameter and w=smaller of perpendiculardiameter.

Statistical Analysis

The statistical analysis of the tumor volume data begins with a datatransformation to a log scale to equalize variance across time andtreatment groups. The log volume data are analyzed with a two-wayrepeated measures analysis of variance by time and treatment using theMIXED procedures in SAS software (Version 9.3). The correlation modelfor the repeated measures is Spatial Power. Treated groups are comparedto the control group at each time point. The MIXED procedure is alsoused separately for each treatment group to calculate adjusted means andstandard errors at each time point. Both analyses account for theautocorrelation within each animal and the loss of data that occurs whenanimals with large tumors are removed from the study early. The adjustedmeans and standard errors (s.e.) are plotted for each treatment groupversus time. Analysis for tumor volume is based on log₁₀ and spatialpower covariance structure. P value is based on the comparison betweentwo specific groups.

Combination Analysis Method (Bliss Independence for IVEF Studies)

First, the usual repeated measures model is fit to log volume versusgroup and time. Then contrast statements are used to test for aninteraction effect at each time point using the 2 specific treatmentsthat are combined. This is equivalent to the Bliss Independence methodand assumes that tumor volumes can, in theory, reach zero, i.e.,complete regression. The expected additive response (EAR) for thecombination is calculated on the tumor volume scale as: response (EAR)EAR volume=V1*V2/V0, where V0, V1, and V2 are the estimated mean tumorvolumes for the vehicle control, treatment 1 alone, and treatment 2alone, respectively. If the interaction test is significant, thecombination effect is declared statistically more than additive or lessthan additive depending on the observed combination mean volume beingless than or more than the EAR volume, respectively. Otherwise, thestatistical conclusion is additive. In addition, a biologically relevantrange of additivity can be defined as X % above and below the EARvolume. Typically, X would be 25 to 40%. Then a biological conclusioncan be made for the combination as more than additive, additive, or lessthan additive if the observed combination mean volume is below, in, orabove the interval of additivity.

There may be situations were stasis is the best expected response. Inthose situations, the Bliss method can be applied directly to the %delta T/C values to obtain an EAR percent response: EAR % deltaT/C=Y1*Y2/100, where Y1 and Y2 are the percent delta T/C values for thesingle-agent treatments. Currently, there is no statistical test tocompare the observed % delta T/C in the combination group versus theEAR, but the biological criterion described above can be applied.

As shown in Table 15 and 16, treatment with Example 1B or abemaciclibalone as a single agent resulted in 32% (% dT/C=−32) and 52% (%dT/C=−52) tumor regressions respectively and both are statisticallysignificant (p<0.001) compared to vehicle control. Combination efficacyof Example 1B with abemaciclib was “Less Than Additive” but thecombination efficacy of Example 1B plus abemaciclib was significantlybetter than Example 1B alone (p<0.001). However, single agentabemaciclib efficacy was not statistically significant from combination(P=0.055). The combination is tolerated in the animals withoutsignificant body weight loss.

TABLE 15 Combination Efficacy of Example 1B with abemaciclib in MCF7 ER-positive breast cancer model Treatment 1 Treatment 2 Difference^(b) SEp-value Vehicle, QD × 42, Example 1B, 10 0.628 0.0658 <0.001* PO mpk, QD× 42, PO Vehicle, QD × 42, Abemaciclib, 50 0.479 0.0658 <0.001* PO mpk,QD × 42, PO Vehicle, QD × 42, Example 1B, 10 0.756 0.0658 <0.001* POmpk, QD × 42, PO/ Abemaciclib, 50 mpk, QD × 42, PO Example 1B, 10Example 1B, 10 0.128 0.0658 0.055 mpk, QD × 42, mpk, QD × 42, PO PO/Abemaciclib, 50 mpk, QD × 42, PO Abemaciclib, 50 Example 1B, 10 0.2780.0658 <0.001* mpk, QD × 42, mpk, QD × 42, PO PO/ Abemaciclib, 50 mpk,QD × 42, PO PO ^(b)Difference = Treatment 1 − Treatment 2; *p-value:significant (p < 0.05) SE—Standard error

TABLE 16 Combination Efficacy of Example 1B with abemaciclib in MCF7 ER-positive breast cancer model Delta T/C % or Combination Treatment %Regression p-value Effect Bodyweight Vehicle NA NA Example 1B −32<0.001* Abemaciclib −52 <0.001* Example 1B/ −64 <0.001* Less Than Nosignificant Abemaciclib/ Additive change (Combination) Analysis fortumor volume is based on Log₁₀ and Spatial Power covariance structure.*p-value: significant (p < 0.05); NA: Not applicable Delta T/C % iscalculated when the endpoint tumor volume in a treated group is at orabove baseline tumor volume; and regression % is calculated for tumorvolume below the baseline. The formula is 100 * (T − T₀)/(C − C₀), whereT and C are mean endpoint tumor volumes in the treated or control group,respectively. T₀ and C₀ are mean baseline tumor volumes in those groups.

As shown in Tables 17 and 18, treatment with Example 1B or Compound Aalone as a single agent resulted in 32% (% dT/C=−32) and 36% (%dT/C=−36) tumor regressions respectively and both are statisticallysignificant (p<0.001) compared to vehicle control. Combination efficacyof Example 1B with Compound A was “Less Than Additive” but thecombination efficacy of Example 1B plus Compound A was significantlybetter than Example 1B alone (p<0.001) or Compound A alone (p=0.002*).The combination is tolerated in the animals without significant bodyweight loss.

TABLE 17 Combination Efficacy of Example 1B with Compound A in MCF7 ER-positive breast cancer model Treatment 1 Treatment 2 Difference^(b) SEp-value Vehicle, QD × 42, Example 1B, 10 0.479 0.0759 <0.001* PO mpk, QD× 42, PO Vehicle, QD × 42, Compound A, 7.5 0.504 0.0759 <0.001* PO mpk,BID × 42, PO Vehicle, QD × 42, Example 1B, 10 0.761 0.0791 <0.001* POmpk, QD × 42, PO/Compound A, 7.5 mpk, BID × 42, PO Example 1B, 10Example 1B, 10 0.282 0.0791 <0.001* mpk, QD × 42, mpk, QD × 42, POPO/Compound A, 7.5mpk, BID × 42, PO Compound A, 7.5 Example 1B, 10 0.2570.0791 0.002* mpk, BID × 42, mpk, QD × 42, PO PO/Compound A, 7.5 mpk,BID × 42, PO PO ^(b)Difference = Treatment 1 − Treatment 2; *p-value:significant (p < 0.05) SE—Standard error

TABLE 18 Combination Efficacy of Example 1B with abemaciclib in MCF7 ER-positive breast cancer model Delta T/C % or Combination Treatment %Regression p-value Effect Bodyweight Vehicle NA NA Example 1B −32<0.001* Compound A −36 <0.001* Example 1B/ −65 <0.001* Less Than Nosignificant Compound A/ Additive change (Combination) Analysis for tumorvolume is based on Log₁₀ and Spatial Power covariance structure.*p-value: significant (p < 0.05); NA: Not applicable Delta T/C % iscalculated when the endpoint tumor volume in a treated group is at orabove baseline tumor volume; and regression % is calculated for tumorvolume below the baseline. The formula is 100*(T − T₀)/(C − C₀), where Tand C are mean endpoint tumor volumes in the treated or control group,respectively. T₀ and C₀ are mean baseline tumor volumes in those groups.

As shown in Tables 19 and 20, treatment with Example 10 or abemaciclibalone as a single agent resulted in 51% (% dT/C=−51) and 70% (%dT/C=−70) tumor regressions respectively and both are statisticallysignificant (p<0.001) compared to vehicle control. Combination efficacyof Example 10 with abemaciclib was “Less Than Additive” but thecombination efficacy of Example 10 plus abemaciclib was significantlybetter than Example 10 alone (p=0.039). However, combination efficacy ofExample 10 plus abemaciclib was not significantly different fromabemaciclib alone (p=0.905). The combination is tolerated in the animalswithout significant body weight loss.

TABLE 19 Combination Efficacy of Example 10 with abemaciclib in MCF7 ER-positive breast cancer model Treatment 1 Treatment 2 Difference^(b) SEp-value Vehicle, Example 10, 15 0.659 0.113 <0.001* QD × 42, PO mpk, QD× 42, PO Vehicle, Abemaciclib 50 0.445 0.1054 <0.001* QD × 42, PO mpk,QD × 42, PO Vehicle, Example 10, 15 0.672 o.1054 <0.001* QD × 42, POmpk, QD × 42, PO/abemaciclib, 50 mpk, QD × 42, PO Example 10, 15 Example10, 15 0.013 0.1103 0.905 mpk, QD × 42, mpk, QD × 42, PO PO/abemaciclib,50 mpk, QD × 42, PO Abemaciclib, 50 Example 10, 15 0.227 0.1054 0.039*mpk, QD × 42, mpk, QD × 42, PO PO/abemaciclib, 50 mpk, QD × 42, PO PO^(b)Difference = Treatment 1 − Treatment 2; *p-value: significant (p <0.05) SE—Standard error

TABLE 20 Combination Efficacy of Example 10 with abemaciclib in MCF7 ER-positive breast cancer model Delta T/C % or Combination Treatment %Regression p-value Effect Bodyweight Vehicle NA NA Example 10 −51<0.001* Abemaciclib −70 <0.001* Example 10/ −71 <0.001* Less Than Nosignificant Abemaciclib/ Additive change (Combination) Analysis fortumor volume is based on Log₁₀ and Spatial Power covariance structure.*p-value: significant (p < 0.05); NA: Not applicable Delta T/C % iscalculated when the endpoint tumor volume in a treated group is at orabove baseline tumor volume; and regression % is calculated for tumorvolume below the baseline. The formula is 100*(T − T₀)/(C − C₀), where Tand C are mean endpoint tumor volumes in the treated or control group,respectively. T₀ and C₀ are mean baseline tumor volumes in those groups.

As shown in Table 21 and 22, treatment with Example 10 or alpelisibalone as a single agent resulted in 51% (% dT/C=−51) and 21% (%dT/C=−21) tumor regressions respectively and both are statisticallysignificant (p<0.001 and p=0.013) compared to vehicle control.Combination efficacy of Example 10 with alpelisib was “Additive” and thecombination efficacy of Example 10 plus alpelisib was significantlybetter than Example 10 alone (p=0.009). Combination efficacy of Example10 plus alpelisib was also significantly better than alpelisib alone(p=<0.001). The combination is tolerated in the animals withoutsignificant body weight loss.

TABLE 21 Combination Efficacy of Example 10 with alpelisib in MCF7ER-positive breast cancer model Treatment 1 Treatment 2 Difference^(b)SE p-value Vehicle, Example 10, 15 0.241 0.1121 <0.039* QD × 42, PO mpk,QD × 42, PO Vehicle, alpelisib 15 mpk 0.445 0.1121 <0.001* QD × 42, PO(d1-d7,), 10 mpk (d8-42), mpk, QD × 42, PO Vehicle, Example 10, 15 0.7550.1121 <0.001* QD × 42, PO mpk, QD × 42, PO/alpelisib 15 mpk (d1-d7,),10 mpk (d8-42), mpk, QD × 42, PO Example 10, 15 Example 10, 15 0.5140.1121 <0.001 mpk, QD × 42, mpk, QD × 42, PO PO/alpelisib 15 mpk(d1-d7,), 10 mpk (d8-42), mpk, QD × 42, PO alpelisib 15 mpk Example 10,15 0.310 0.1121 0.009* (d1-d7,), 10 mpk mpk, QD × 42, (d8-42), mpk,PO/alpelisib 15 QD × 42, PO mpk (d1-d7,), 10 mpk (d8-42), mpk, QD × 42,PO ^(b)Difference = Treatment 1 − Treatment 2; *p-value: significant (p< 0.05) SE—Standard error

TABLE 22 Combination Efficacy of Example 10 with alpelisib in MCF7ER-positive breast cancer model Delta T/C % or Combination Treatment %Regression p-value Effect Bodyweight Vehicle NA NA Example 10 −51<0.001* alpelisib −21 <0.013* Example 10/ −76 <0.001* Additive Nosignificant alpelisib change (Combination) Analysis for tumor volume isbased on Log₁₀ and Spatial Power covariance structure. *p-value:significant (p < 0.05); NA: Not applicable Delta T/C % is calculatedwhen the endpoint tumor volume in a treated group is at or abovebaseline tumor volume; and regression % is calculated for tumor volumebelow the baseline. The formula is 100*(T − T₀)/(C − C₀), where T and Care mean endpoint tumor volumes in the treated or control group,respectively. T₀ and C₀ are mean baseline tumor volumes in those groups.

As shown in Table 23 and 24, treatment with Example 10 or everolimusalone as a single agent resulted in 51% (% dT/C=−51) and 50% (%dT/C=−50) tumor regressions respectively and both are statisticallysignificant (p<0.001 and p<0.001) compared to vehicle control.Combination efficacy of Example 10 with everolimus was “Additive” andthe combination efficacy of Example 10 plus everolimus was significantlybetter than Example 10 alone (p=0.004). Combination efficacy of Example10 plus alpelisib was also significantly better than everolimus alone(p=0.04). The combination is tolerated in the animals withoutsignificant body weight loss.

TABLE 23 Combination Efficacy of Example 10 with everolimus in MCF7 ER-positive breast cancer model Treatment 1 Treatment 2 Difference^(b) SEp-value Vehicle, QD × 42, Example 10, 15 0.445 0.0999 <0.001* PO mpk, QD× 42, PO Vehicle, QD × 42, Everolimus, 5 0.433 0.1038 <0.001* PO mpk, QD× 42, PO Vehicle, QD × 42, Example 10, 15 0.748 0.0999 <0.001* PO mpk,QD × 42, PO/Everolimus, 5 mpk, QD × 42, PO Example 10, 15 Example 10, 150.303 0.0999 0.004* mpk, QD × 42, mpk, QD × 42, PO PO/Everolimus, 5 mpk,QD × 42, PO Everolimus, 5 Example 10, 15 0.315 0.138 0.004* mpk, QD ×42, mpk, QD × 42, PO PO/Everolimus, 5 mpk, QD × 42, PO ^(b)Difference =Treatment 1 − Treatment 2; *p-value: significant (p < 0.05) SE—Standarderror

TABLE 24 Combination Efficacy of Example 10 with everolimus in MCF7 ER-positive breast cancer model Delta T/C % or Combination Treatment %Regression p-value Effect Bodyweight Vehicle NA NA Example 10 −51<0.001* Everolimus −50 <0.001* Example 10/ −76 <0.001* Additive Nosignificant Everolimus change (Combination) Analysis for tumor volume isbased on Log₁₀ and Spatial Power covariance structure. *p-value:significant (p < 0.05); NA: Not applicable Delta T/C % is calculatedwhen the endpoint tumor volume in a treated group is at or abovebaseline tumor volume; and regression % is calculated for tumor volumebelow the baseline. The formula is 100*(T − T₀)/(C − C₀), where T and Care mean endpoint tumor volumes in the treated or control group,respectively. T₀ and C₀ are mean baseline tumor volumes in those groups.

Rat Oral Bioavailability Assay

The purpose of the following assay is to demonstrate whether a testcompound is orally bioavailable.

Administer the test compound to Sprague-Dawley rats IV at 1 mg/kg (usingvehicles of either: 20% CAPTISOL® in 25 mM sodium phosphate buffer, pH2quantum satis; or 25% DMA, 15% EtOH, 10% propylene glycol, 25%2-pyrrolidone, and 25% purified water) and PO at 10 mg/kg (using avehicle of 1% hydroxyethyl cellulose, 0.25% polysorbate 80, 0.05%Antifoam 1510-US, and purified water quantum satis). Collect serialblood samples at 0.08, 0.25, 0.5, 1, 2, 4, 8, and 12 hours post dose forIV bolus and at 0.25, 0.5, 1, 2, 4, 8, and 12 hours post dose after oraladministration. After treatment with an EDTA coagulant, obtain plasma bycentrifugation and store at −70° C. until analysis by LC-MS/MS.Determine the test compound concentration in plasma and upload into theWatson LIMS™ system where noncompartmental analysis is used to calculateArea Under the Curve (AUC) for both IV and PO arms. Calculate oralbioavailability (% F) via the following equation,

% F=(AUC_(PO)λDose_(IV))/(AUC_(IV)×Dose_(PO))×100.

The compounds of Example 1B displays a % F value of ˜50% in theabove-mentioned assay. This assay demonstrates that Example 1B has goodoral bioavailability.

We claim:
 1. A compound of the formula:

wherein either R¹ or R² is independently selected from Cl, F, —CF₃, or—CH₃, and the other is hydrogen, or a pharmaceutically acceptable saltthereof.
 2. The compound according to claim 1, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 3. The compound accordingto claim 1, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 4. The compound accordingto claim 1, wherein the pharmaceutically acceptable salt is abenzenesulfonic acid salt.
 5. The compound according to claim 1, whereinthe pharmaceutically acceptable salt is a 4-methylbenzenesulfonic acidsalt.
 6. The compound according to claim 2, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 7. The compound accordingto claim 6, wherein the pharmaceutically acceptable salt is abenzenesulfonic acid salt.
 8. The compound according to claim 6, whereinthe pharmaceutically acceptable salt is a 4-methylbenzenesulfonic acidsalt.
 9. The compound according to claim 6, wherein the compound is


10. The compound according to claim 3, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 11. The compoundaccording to claim 10, wherein the pharmaceutically acceptable salt is abenzenesulfonic acid salt.
 12. The compound according to claim 10,wherein the pharmaceutically acceptable salt is a4-methylbenzenesulfonic acid salt.
 13. The compound according to claim10, wherein the compound is


14. A pharmaceutical composition comprising the compound or thepharmaceutically acceptable salt thereof according to claim 1 incombination with a pharmaceutically acceptable excipient, carrier, ordiluent.
 15. The pharmaceutical composition according to claim 14,comprising one or more other therapeutic agents.
 16. A method oftreating breast cancer, ovarian cancer, endometrial cancer, prostatecancer, uterine cancer, gastric cancer, or lung cancer, comprisingadministering to a patient in need of such treatment an effective amountof a compound or a pharmaceutically acceptable salt thereof according toclaim
 1. 17. The method according to claim 16, wherein the breast canceris ER-positive breast cancer.
 18. The method according to claim 16,wherein the gastric cancer is ER-positive gastric cancer.
 19. The methodaccording to claim 16, wherein the lung cancer is ER-positive lungcancer.